Lysis sensing receptors and uses thereof

Abstract

Disclosed herein are cells (e.g, immune cells such as T cells) engineered to contain and / or express a Lysis Sensor Receptors (LSR) comprising an extracellular domain with affinity and specificity for an intracellular product of a diseased cell such as a cancer cell; a transmembrane element and at least one signaling element, and a chimeric antigen receptor (CAR) comprising an extracellular domain with affinity and specificity for an extracellular product of a diseased cell such as a cancer cell; a transmembrane element and at least one signaling element. Also provided herein are various uses of such engineered cells, including in the treatment of cancer, for example to enhance T cell activity (e.g, TIL and / or CAR-T cell activity) and / or to enhance antibody-based therapy, such as cancer immunotherapy.

Description

Docket No.: 2017422-0007 LYSIS SENSING RECEPTORS AND USES THEREOF BACKGROUND

[0001] Cancer immunotherapy, including CAR-T cell therapy, is used to provokeimmune responses attacking tumor cells while sparing normal tissues. The present disclosure provides technologies related to immune cells comprising a lysis sensing CAR targeting an intracellular product of tumor cells and related methods. SUMMARY

[0002] Targeted immunotherapies rely on the use of immune cells or molecules that engage immune cells to treat a variety of diseases, including primarily cancer but also infectious diseases and autoimmune disorders. Recently, engineering T cells to express chimeric antigen receptors (CARs) that target tumor antigens has allowed the successful eradication of leukemic cells in humans. CARs have two functional elements: an extracellular antigen recognition element, typically derived from an antibody, and an intracellular stimulatory element or set of elements. Upon recognition of an antigen on a tumor cell, CARs activate cytotoxic activity of T cells, which in turn results in tumor cell lysis.

[0003] Among other things, the present disclosure identifies challenges with certain existing CAR-T cell therapies, including, for example that an antigen must be expressed on a tumor cell surface to be available for recognition. Alternatively or additionally, the present disclosure identifies challenges with existing CAR-T cell therapies in that most tumor cell surface antigens that have been identified or utilized as targets of such CAR-T cell therapies are also expressed on healthy cells, reducing tolerance to CAR-T therapy and limiting effectiveness.

[0004] The present disclosure appreciates that many epitopes preferentially or specifically associated with tumors (e.g., neoantigen epitopes, and many epitopes associated with tumor driver mutations) are primarily or wholly intracellular. Although intracellular proteins can be recognized by T cell receptors (TCRs) if presented to them, that presentation process (a) is not specific to tumor-associated (let alone tumor-specific) antigens; and (b) presents only small fragments of intracellular proteins, which may not include relevant Page 1 of 89 12923515v1Docket No.: 2017422-0007 epitopes (e.g., that are tumor-associated or tumor-specific). Thus, only small fragments of these proteins are presented to TCRs. This, in turn, limits the ability of the immune system to detect intracellular mutations. Furthermore, as tumors accumulate mutations, the neoantigens that they present to T cells change, rendering some previously responsive T cells ineffective.

[0005] The present disclosure provides an insight that connecting T cell activation to presence of lysed / lysing cells could dramatically improve engineered T cell therapy (e.g., CAR-T therapy). Among other things, the present disclosure provides CAR constructs that respond specifically to cell lysis, described herein as a Lysis Sensor Receptor (LSR). The present disclosure describes LSR constructs comprising, for example, an extracellular lysis- antigen-binding moiety, a transmembrane element, and an intracellular T cell receptor co- stimulation element. The present disclosure further provides engineered immune cell populations (e.g., engineered T cells) that express such LSRs, for example as a Co-CAR receptor. The present disclosure further provides various insights and technologies relating to such LSR constructs and / or their expression by engineered cells including, for example, methods of making and / or using such constructs and / or cells that express them.

[0006] Among other things, the present disclosure provides an insight that immune cells expressing such a LSR construct may be useful to enhance immune responses (e.g., by activating endogenous T cells), for example in the treatment of cancer. Among other things, the present disclosure provides an insight that immune cells expressing such a LSR construct may be useful to enhance immune responses (e.g., by activating endogenous T cells), for example in the treatment of solid tumors. LSRs provide a mechanism to dramatically expand the universe of antigens that can be used to stimulate an immune response in diseased tissues by bypassing the limitations of other immunotherapies imposed by the intracellular location of potential target antigens.

[0007] The present disclosure recognizes that LSRs have a further advantage over other activators of the immune response in that their activating antigens are only made available after cell lysis, thereby reducing the risk of toxicities that can result from antigens which may be expressed on the surface of both healthy and diseased cells. In some embodiments, LSRs are designed to selectively bind to antigens that are disease-specific, such as cancer- specific mutants or viral proteins, further reducing the potential for inadvertent immune cell Page 2 of 89 12923515v1Docket No.: 2017422-0007 activation by processes involved in healthy cell turnover. Dependence on cell lysis serves as a positive feedback loop which increases the activity of immune cells in diseased tissues while minimizing their activation in other areas of the body.

[0008] The present disclosure describes that populations of T cells (e.g., a population of T cells isolated from a human) engineered to express a LSR maintain their endogenous TCR heterogeneity. Thus, in some embodiments, engineered T cell subpopulations will recognize diseased cells even if disease-specific antigens presented to TCR subclones change.

[0009] In some embodiments, LSRs will amplify the response of any T cells in the vicinity of a diseased cell that are capable of recognizing a disease-specific antigen, thereby stimulating cytotoxicity and reinforcing clonal expansion of T cells with effective TCRs. This approach provides a distinct advantage in the treatment of diseases with rapidly changing characteristics, such as tumor cells or viral infections, by limiting the need for artificial amplification of tumor antigen presentation or genetic engineering to customize LSRs for specific antigens. As a tumor (or other diseased tissue or cell, such as infected cells) evolves and presentation of antigens changes, LSRs will stimulate T cells encoding TCRs that recognize these new antigens without the need to develop a new therapy and thereby limiting the potential for resistance to develop.

[0010] In some embodiments, use of LSRs as described herein can provide immune cell therapies, e.g., for solid tumors, that are (i) more effective, (ii) safer and (iii) more durable than existing cell therapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawing, comprising the below-described figures, forms part of the present specification and is included to further demonstrate and / or facilitate appreciation of certain aspects of the present disclosure.

[0012] FIG.1 shows an image depicting a positive feedback loop between activated engineered T cells expressing a LSR reacting with and lysing tumor cells in their proximity causing lysis of more tumor cells, resulting in more activation of LSR-expressing T cells.

[0013] FIG.2 shows an image depicting certain exemplary LSR constructs comprising an extracellular single chain variable fragment (scFv) operably linked to a transmembrane Page 3 of 89 12923515v1Docket No.: 2017422-0007 element, and one or more T cell co-stimulation elements. In some embodiments, an exemplary LSR construct further comprises a CD3^ co-stimulation element.

[0014] FIGs.3A-3E show images depicting exemplary models for LSR activation by a lysis-associated-antigen (e.g., antigens that are made available to a LSR upon the lysis of a diseased cell (e.g., intracellular antigens / epitopes)).

[0015] FIG.4 shows an image depicting exemplary anti-KRAS LSR constructs.

[0016] FIG.5 shows an image depicting activation and oligomerization of LSRs by KRAS binding.

[0017] FIG.6 shows a table depicting a number of oncogenes mutated in a high percentage of tumors; several of the proteins encoded by these oncogenes are intracellular proteins. The present disclosure provides LSR constructs (and associated technologies, as described herein) that target such intracellular oncogene-encoded proteins.

[0018] FIG.7 shows an image depicting an exemplary Anti-P53 LSR construct.

[0019] FIG.8 shows an image depicting exemplary steps of a LSR T therapy.

[0020] FIG.9 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Abecma (idecabtagene vicleucel).

[0021] FIG.10 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Breyanzi (lisocabtagene maraleucel).

[0022] FIG.11 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Kymriah (tisagenlecleucel).

[0023] FIG.12 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Tecartus (brexucabtagene autoleucel).

[0024] FIG.13 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Yescarta (axicabtagene ciloleucel).

[0025] FIG.14 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Carvykti (ciltacabtagene autoleucel). Page 4 of 89 12923515v1Docket No.: 2017422-0007

[0026] FIG.15 depicts two exemplary LSR construct designs according to an embodiment of the present disclosure.

[0027] FIG.16 depicts flow cytometry data indicating detection of KRAS-LSR-T cells expressing GFP and a KRAS-LSR construct.

[0028] FIG.17 depicts flow cytometry data indicating detection of KRAS-LSR-T cells expressing GFP and a KRAS-LSR construct, Meso-CAR-T cells, and dual T cells expressing both a Meso-CAR and a KRAS-LSR (e.g., dual KRAS-LSR / Meso-CAR-T cell).

[0029] FIG.18 shows a graph depicting percent cell killing of A1847 cells by dual KRAS-LSR / Meso-CAR-T cells, Meso-CAR-T cells, and KRAS-LSR-T cells over time (0 to 21 hours).

[0030] FIG.19 shows a schematic depicting a proposed mechanism of action for increased T cell activation and increased cell killing by an exemplary KRAS LSR.

[0031] FIG.20 shows a graph depicting flow cytometry data depicting detection of KRAS-LSR-T cells expressing GFP and a KRAS-LSR construct, EpCAM-CAR-T cells, and dual T cells expressing both a EpCAM-CAR and a KRAS-LSR (e.g., dual KRAS- LSR / EpCAM-CAR-T cell).

[0032] FIG.21 shows a graph depicting percent cell killing of SW-1990 cells by dual KRAS-LSR / EpCAM-CAR-T cells, EpCAM-CAR-T cells, and KRAS-LSR-T cells over time (0 to 20 hours).

[0033] FIG.22 shows a graph depicting IFN-gamma levels in supernatant collected from A1847 cells (e.g., human ovarian cancer cells) cultured with Meso-CAR-T cells, Meso- CAR / KRAS-LSR T cells (e.g., T cells expressing both a KRAS-targeting LSR and a Meso- CAR, or non-transduced T cells. Meso-CAR / KRAS-LSR T cells have a higher degree of T cell activation, as measured by IFN-gamma expression, than Meso-CAR-T cells alone.

[0034] FIG.23 shows a graph depicting fractional cell killing of A1847 cells by KRAS- LSR-T cells, Meso-CAR-T cells, a mixture of KRAS-LSR-T cells and Meso-CAR-T cells, and dual KRAS-LSR / Meso-CAR-T cells over time (0 to 48 hours). Page 5 of 89 12923515v1Docket No.: 2017422-0007

[0035] FIG.24 shows a graph depicting fractional cell killing of A1847 cells by KRAS- LSR-T cells with addition of purified KRAS protein or supernatant from A1847 cells previously killed by Meso-CAR-T cells. Addition of supernatant from A1847 cells previously killed by Meso-CAR-T cells, resulted in moderate cell killing of A1847 cells by KRAS-LSR-T cells. Addition of purified G12D KRAS resulted in increased killing of A1847 cells by KRAS-LSR-T cells relative to addition of supernatant from A1847 cells previously killed by Meso-CAR-T cells.

[0036] FIG.25 shows a graph depicting fractional cell killing of A1847 cells by dual KRAS-LSR / Meso-CAR-T cells with and without addition of an anti-KRAS antibody. Addition of anti-KRAS antibody reduced rate of cell killing by dual KRAS LSR / Meso- CAR-T cells.

[0037] FIG.26 shows a schematic depicting a proposed mechanism of action for T cell activation by a tumor antigen and priming by a lysis-associated antigen and specific cell killing of tumor cells by an exemplary zlessCAR / LSR described herein.

[0038] FIG.27 shows two exemplary dual expression CAR / LSR construct designs according to an embodiment of the present disclosure.

[0039] FIG.28 shows T cells co-expressing a zlessMeso-CAR and a KRAS LSR (i.e., zlessMeso-CAR / KRAS-LSR-T cells), and T cells co-expressing a Meso-CAR and a KRAS LSR (i.e., Meso-CAR / KRAS-LSR-T cells) (KRAS-LSR: anti-Flag-APC; Meso-CAR: Mesothelin-FITC). T cells were produced using dual expression CAR / LSR construct designs described herein.

[0040] FIG.29 shows a graph depicting IFN-gamma levels in supernatant collected from zlessMeso-CAR / KRAS-LSR-T cells incubated with mutant and wild-type KRAS proteins.

[0041] FIG.30 shows a graph depicting fractional cell killing of A1847 cells over time by Meso-CAR-T cells (10:1), a combination of Meso-CAR-T cells (1:1) and zlessMeso- CAR-T cells (10:1), a combination of Meso-CAR-T cells (1:1) and KRAS-LSR-T cells (10:1), Meso-CAR-T cells (1:1), zlessMeso-CAR-T cells (10:1), and KRAS-LSR-T cells Page 6 of 89 12923515v1Docket No.: 2017422-0007 (10:1). A KRAS-LSR did not show cell killing activity when expressed independently from a Meso-CAR. zlessMeso-CAR-T cells did not show cell killing activity.

[0042] FIG.31 shows a graph depicting relative target cell number over time after incubation with Meso-CAR-T cells (1:10), zlessMeso-CAR / KRAS-LSR-T cells (3:1), a combination of zlessMeso-CAR / KRAS-LSR-T cells (3:1) and Meso-CAR (1:10), Meso- CAR-T cells (1:1), and a combination of zlessMeso-CAR / KRAS-LSR-T cells (3:1) and Meso-CAR-T cells (1:1).

[0043] FIG.32 shows a bar graph depicting relative amount of IFN-gamma secreted after 25 hours incubation of SW1116 or ASPC-1 cells with either Meso-CAR-T cells alone, zlessMeso-CAR / KRAS-LSR-T cells alone, or a combination of zlessMeso-CAR / KRAS- LSR-T cells with priming Meso-CAR-T cells. DEFINITIONS

[0044] Administration: As used herein, the term “administration” typically refers to the administration (e.g., of a composition or treatment) to a subject or system (e.g., that is or comprises one or more cells, tissues, organisms, etc.), for example to achieve delivery of an agent that is, is included in, or is otherwise delivered or generated by, such composition or treatment.

[0045] CDR: as used herein, the term “CDR” refers to a complementarity determining region within an immunoglobulin (e.g., antibody, T cell receptor) variable region. Those skilled in the art are aware that canonical antibodies include heavy and light chains, each of which is comprised of a variable region and a constant region. There are three CDRs in each such variable region, designated CDR1, CDR2 and CDR3. Analogously, T cell receptors comprise a and B chains, or d and g chains, each of which includes 3 CDRs. A "set of CDRs" or "CDR set" refers, as will be clear from context and understood by those skilled in the art, either to the group of three CDRs that occur in a particular chain, or to the set of six CDRs (i.e., three heavy chain and three light chain CDRs) that are found together in a particular immunoglobulin (and e.g., contribute to or determine its antigen-specificity). Certain systems have been established in the art for defining CDR boundaries (e.g., Kabat, Chothia, etc.); those skilled in the art appreciate the differences between and among these Page 7 of 89 12923515v1Docket No.: 2017422-0007 systems and are capable of understanding CDR boundaries to the extent required to understand and to practice the claimed invention.

[0046] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0047] Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, Page 8 of 89 12923515v1Docket No.: 2017422-0007 CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure.

[0048] Designed: As used herein, the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and / or (iii) that is distinct from natural substances and other known agents.

[0049] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0050] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing Page 9 of 89 12923515v1Docket No.: 2017422-0007 regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0051] Effector function: as used herein refers a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include but are not limited to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). In some embodiments, an effector function is one that operates after the binding of an antigen, one that operates independent of antigen binding, or both.

[0052] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. For example, in some embodiments described and / or utilized herein, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and / or if produced other than natural expression in a cell. Analogously, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and / or expresses a Page 10 of 89 12923515v1Docket No.: 2017422-0007 particular agent of interest (e.g., a protein, a nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0053] Framework" or "framework region: as used herein, refers to the sequences of an immunoglobulin (e.g., antibody) variable region minus the CDRs. Because a CDR sequence can be determined by different systems, likewise a framework sequence is subject to correspondingly different interpretations. The six CDRs divide the framework regions on the heavy and light chains into four sub-regions (FRl, FR2, FR3 and FR4) on each chain, in which CDRl is positioned between FRl and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, FR1, for example, represents the first framework region closest to the amino terminal end of the variable region and 5' with respect to CDR1, and FRs represents two or more of the sub-regions constituting a framework region.

[0054] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution. Typical amino acid categorizations are summarized below: Page 11 of 89 12923515v1Docket No.: 2017422-0007 Alanine Ala A nonpolar neutral 1.8 Arinine Ar R olar ositive -45    Ambiguous Amino Acids 3-Letter 1-LetterPage 12 of 89 12923515v1Docket No.: 2017422-0007 Ambiguous Amino Acids 3-Letter 1-Letter Leucine or Isoleucine Xle J [ ap p q g homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences. Calculation of the percent homology between two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that position. The percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Representative algorithms and computer programs useful in determining the percent homology between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent homology between two nucleotide sequences can, alternatively, be determined for example using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0056] Identity: As used herein, the term “identity” refers to overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or Page 13 of 89 12923515v1Docket No.: 2017422-0007 RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, a length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of length of a reference sequence; residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as a corresponding position in the second sequence, then the two molecules (i.e., first and second) are identical at that position. Percent identity between two sequences is a function of the number of identical positions shared by the two sequences being compared, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17, which is herein incorporated by reference in its entirety), which has been incorporated into the ALIGN program (version 2.0). In some embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0057] “Improve,” “increase”, “inhibit” or “reduce”: As used herein, the terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement Page 14 of 89 12923515v1Docket No.: 2017422-0007 may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0058] Isolated: as used herein, refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting) and / or otherwise previously associated, and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. In some embodiments, a substance may be considered to be “isolated” if it is (or has been caused to be) free of or separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components (e.g., components with which it was previously associated). In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be "isolated" when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components a) with Page 15 of 89 12923515v1Docket No.: 2017422-0007 which it is associated in nature; and / or b) with which it was associated when initially produced.

[0059] Operably linked: as used herein, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, "operably linked" control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a from the functional element of interest.

[0060] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0061] Prevent or prevention: as used herein when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. In some embodiments, prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0062] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized Page 16 of 89 12923515v1Docket No.: 2017422-0007 under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0063] Specific binding: As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. A binding agent that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree and / or rate of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree and / or rate of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.

[0064] Susceptible to: An individual who is “susceptible to” a disease, disorder, and / or condition is one who has a higher risk of developing the disease, disorder, and / or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0065] Tumor: As used herein, the term “tumor” refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, a tumor is associated with, or is a manifestation of, a cancer. In some Page 17 of 89 12923515v1Docket No.: 2017422-0007 embodiments, a tumor may be a disperse tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor.

[0066] Tumor-specific: As used herein, the term “tumor-specific” refers to an agent that is associated with tumor cells and not with appropriate reference non-tumor cells. In some embodiments, neoantigens (e.g., that arise in a tumor, for example by mutation that occurs in the tumor) are tumor-specific antigens.

[0067] Tumor-associated: As used herein, the term “tumor-associated” refers to an agent that is more likely to be present (or detectable) in and / or on tumor cells than appropriate reference non-tumor cells.

[0068] Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and / or contributing to a particular structural motif and / or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three- dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with Page 18 of 89 12923515v1Docket No.: 2017422-0007 a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature. In some embodiments, a reference polypeptide or nucleic acid is a human polypeptide or nucleic acid. Page 19 of 89 12923515v1Docket No.: 2017422-0007

[0069] Vector: as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Adoptive Cell Therapy

[0070] Adoptive Cell Therapy (ACT) strategies are revolutionizing cancer care by using immune cells – typically cultured immune cells as therapeutic agents to attack patients’ tumors. Most ACT therapies can be grouped into one of three types: (i) tumor-infiltrating lymphocyte (TIL) therapy, a T-cell receptor therapy designed to boost activity of naturally occurring immune cells found in a patient’s tumor, (ii) TCR cell therapy which relies on creating T cells enriched with specific T-cell receptors (TCR) to mediate tumor targeting, and (iii) chimeric antigen receptor (CAR) therapy, which instead utilizes an engineered CAR construct to target T cells (reviewed, for example, in Rosenberg & Restifo, Science 348:62, Page 20 of 89 12923515v1Docket No.: 2017422-0007 2015; June et al., Science 359:1361, 2018; etc., the contents of which is hereby incorporated by reference herein in its entirety). Tumor-Infiltrating Lymphocyte (TIL) Therapy

[0071] Initial adoptive cell therapies utilized tumor infiltrating lymphocytes (TIL) that were isolated from patients’ tumors. Although targets of these TIL are not known, it is presumed that TIL isolated from a tumor are enriched in T cells directed against cancer cells. These TIL are typically expanded and activated ex vivo, and optionally selected or purified to enrich for cells demonstrated to target antigens expressed by tumors. These tumor- associated or tumor-specific antigens, can include antigens associated with tumor drivers, neoantigens (novel antigens arising from genetic mutations) or other antigens that are enriched on tumor cells compared to healthy cells). An expanded population of TIL is then administered to a patient, who is usually the same person from whom the original TIL were isolated. TIL therapy was first demonstrated to achieve regression in melanoma patients in 1988 (see, Rosenberg et al., N. Engl. J. Med 319:1676, 1988, the contents of which is hereby incorporated by reference herein in its entirety), but initial responses were short-lived and administered T-cells were undetectable in the recipients within just a few days. It was subsequently determined that lymphoablation prior to administration of a TIL preparation could dramatically improve durability of response, and apparent successful engraftment of administered TIL (see, for example, Dudley et al., Science 298:850, 2002, the contents of which is hereby incorporated by reference herein in its entirety). Lymphoablation can be utilized prior to any adoptive cell therapy. More recently, improvements in TIL therapy have been driven by use of certain factors (e.g., cytokines, certain antibodies, such as certain anti- PD-1 and / or anti-CTLA-4 antibodies, etc.) to enhance survival and activation of TIL (see, Zhao and Cao, Front Immunol 2019;10:2250, the contents of which is hereby incorporated by reference herein in its entirety). In some embodiments one or more such agents (e.g., in certain embodiments one or more cytokines) are added exogenously. In some embodiments, TIL are genetically engineered to express one or more such agents (e.g., one or more cytokines). Page 21 of 89 12923515v1Docket No.: 2017422-0007 T-cell Receptors (TCR) Therapy

[0072] TCR therapies, which include both naturally occurring TILs and engineered cells involve administering T cells whose T-cell receptors (TCRs) target particular antigens of interest (e.g., tumor antigen, such as a tumor-associated antigen or a tumor-specific antigen). T-cell receptors are transmembrane heterodimers of either ^- and ^- chains or ^- and ^- chains containing CDRs that together determine characteristics of the receptor’s antigen binding (e.g., its specificity and / or affinity, etc.). Cells in the body have a mechanism for displaying on their surface a sampling of every protein that is being made by a cell. This includes all normal proteins as well as aberrant proteins if a cell is cancerous or proteins from pathogens if a cell has been infected. These proteins are broken down into short fragments, or peptides, and these peptides are loaded into Major Histocompatibility Complexes, or MHCs, to be displayed on the outside of a cell. TCRs recognize their cognate antigens only when such antigens are presented by the MHC.

[0073] The specific makeup of an individual’s MHC is determined by their Human Leukocyte Antigen, or HLA, type. For a TCR to recognize an antigen, it should match both the HLA type expressed by cells in that patient and a specific antigen peptide. It has been estimated that there are 1010(10 billion) distinct TCRs in an individual providing the opportunity for recognition of a broad range of potential antigens (see, J Theor Biol.2016 Jan 21; 389: 214–224, the contents of which is hereby incorporated by reference herein in its entirety). However, within a given individual these TCRs are understood to recognize a specific HLA type. In cases where T cells have been engineered with TCRs designed to target select tumor-specific antigens, their application is limited to individuals who also have a matching HLA type. Chimeric Antigen Receptor (CAR) Therapy

[0074] CAR-T therapy involves engineering T cells to express a chimeric antigen receptor (CAR) construct, to enable T cells to be activated independent of a T cell receptor. Such a CAR construct links an antigen binding moiety, typically a single chain variable fragment (scFv) of an antibody known to bind the relevant antigen of interest, with TCR signaling domain(s) and / typically, one or more additional T cell co-stimulation elements (e.g., derived from receptors such as CD28, OX40, CD137, etc.). CAR constructs target Page 22 of 89 12923515v1Docket No.: 2017422-0007 surface antigens, and typically do so with very high avidity. A limitation of CAR-T therapies is that CARs cannot penetrate cells and thereby cannot recognize intracellular antigens. CAR-T therapy is also distinguished from TCR therapy through its ability to recognize antigens independent of HLA type. However, in practice, most CAR-T therapies are generated as autologous therapies to limit potential for mismatches in HLA type that can arise when generating CAR-T therapies from allogeneic donors. These mismatches can lead to the rejection of therapy.

[0075] The United States Food and Drug Administration has approved at least six adoptive cell therapies, all of which are CAR-T therapies, approved for treatment of hematological cancers. Specifically, as of November 2022, CAR-T therapies have been approved for the treatment of large B-cell lymphoma , multiple myeloma, follicular lymphoma, mantle cell lymphoma and acute lymphoblastic leukemia. Certain approved CAR-T therapies have reported stunning success, including recurrence rates reduced by 80% or more. However, cytokine release syndrome (also known as “cytokine storm” has plagued certain CAR-T therapies, which seem to induce much more dramatic such reactions than have been observed with TCR (e.g., TIL and / or engineered TCR) therapies (see, for example, Kalos et al., Sci. Transl. Med.3:95ra73, 2011, the contents of which is hereby incorporated by reference herein in its entirety).

[0076] Upon administration to patients, activation of potent CAR-T therapies often leads to the production of a large number of inflammatory cytokines, a condition known as cytokine release syndrome (CRS). CRS can be a life-threatening consequence of CAR-T therapy leading to modifications of treatment regimens to limit its severity and to the development of CAR-T therapies with safety switches which lead to the inactivation of the therapy with the intent of halting the escalation of CRS (see, Zhao and Cao, Front Immunol 2019;10:2250, the contents of which is hereby incorporated by reference herein in its entirety).

[0077] CAR-T therapies that have been approved by the FDA have exclusively targeted antigens that are exposed on proteins expressed on a cell surface. These therapies have been primarily focused on common surface proteins that are expressed on many hematologic malignancies. By contrast, solid tumors have a high degree of heterogeneity with few common surface-expressed antigens which limits the potential of generating broadly Page 23 of 89 12923515v1Docket No.: 2017422-0007 effective CAR-T therapies. In addition to the scarcity of tumor-specific targets, most surface-expressed proteins on tumor cells are also expressed on healthy cells, leading to toxicities due to effects of the highly cytotoxic CAR-T cells in non-tumor tissues. For example, HER2 is a surface-expressed protein that is also expressed in tissues such as the lung. The first patient to be treated with a HER-2 CAR-T cell therapy developed by the National Cancer Institute fell into a coma soon after and died five days later (Zhao and Cao, Front Immunol 2019;10:2250, the contents of which is hereby incorporated by reference herein in its entirety).

[0078] Furthermore, expression of surface-expressed antigens can be lost, resulting in evasion of therapies that target them. An advantage of TIL therapy is that patients are treated with a population of cells that contain distinct TCRs, increasing the probability that the treatment contains cells that can target multiple tumor-specific antigens – antigens that exist at the time of treatment and antigens that may emerge as the tumor cells emerge that have altered antigens (see, Wang et al. BMC Medicine 2021). Due to the dependence of TIL therapy on TCR activation, it can detect protein changes that occur within cells where the majority of tumor-specific changes occur (see, Schietinger et al., 2008, the contents of which is hereby incorporated by reference herein in its entirety).

[0079] A previous study demonstrated that CARs could be created that act as co- stimulatory CARs, or coCARs. Such coCARs have been shown to stimulate cell killing activity of T cells, but the specificity of cell killing was dependent on the ability of T cells to recognize tumor cells using another receptor, such as a TCR. A coCAR targeting CD19, a surface antigen expressed on hematopoietic cells, has been designed by removing the CD3^ (e.g., CD3 zeta, e.g., CD3Z or CD3C) activation element from the CAR construct, creating a coCAR that was incapable of activating T cells on its own. Limiting the activity of a CD19 coCAR prevented CAR-T cells from killing all hematopoietic cells, while stimulating cell killing of tumor cells targeted by specific TCRs (see, Omer et al., Cancer Immunol Res. 2022 Apr 1;10(4):512-524., the contents of which is hereby incorporated by reference herein in its entirety).

[0080] CAR-T cells containing two CARs (e.g., DuoCARs) have been described (see, US 2024 / 0043497 Al, the contents of which is hereby incorporated by reference herein in its entirety). These DuoCARs were proposed to address a problem with CAR-T cells which is a Page 24 of 89 12923515v1Docket No.: 2017422-0007 lack of surface-expressed antigens that are tumor specific. As described herein, most tumor antigens, including those commonly targeted by CAR-T cell therapies such as CD19, CD20, CD22 and BCMA, are also expressed on healthy cells. However, because these antigens are primarily expressed on hematopoietic cells, patients can typically tolerate and recover from adverse effects associated with broad hematopoietic toxicity. This is not the case with CAR- T therapies that have been created to target solid tumors. Toxicities associated with targeting solid tumors with known tumor antigens such as HER2, EGFR and PSMA, have limited the ability to deliver effective doses of CAR-T cells and hampered their development (see, Maalej et al., Mol Cancer.2023 Jan 30;22(1):20, the contents of which is hereby incorporated by reference herein in its entirety).

[0081] In a DuoCAR strategy, activation of CAR-T cells requires engagement of two separate antigens. Through such approach, a higher degree of specificity for tumor cells is proposed. Dual targeting of DuoCARs is driven through engineering of each of the two CARs. One CAR is created with a high affinity binding domain to a specific tumor antigen, but this CAR does not contain a CD3^ activation element and is thus inactive on its own. A second CAR is created with a lower affinity antigen binding domain to an independent tumor antigen and this CAR contains a CD3^ activation element. Binding of a high affinity CAR to a tumor brings a lower affinity CAR in proximity to its antigen and combined binding of both CARs leads to CAR-T activation. On a molecular level, the co-stimulatory domains of the two CARs act in trans, with neither of the CARs potent enough to trigger T cell activation on their own.

[0082] Among other things, it is an insight of the present disclosure that coCARs are particularly useful in combination with a lysis sensing receptor (LSR) to specifically target solid tumors. In some embodiments, the present disclosure provides cells (e.g., immune cells, e.g., T cells) engineered to express a lysis sensor receptor (LSR) comprising a lysis- associated-antigen binding moiety, a transmembrane element, and a signaling element (e.g., a T cell co-stimulation element or elements) and a chimeric antigen receptor (CAR) comprising an antigen-binding moiety, a transmembrane element, and a signaling element, wherein the signaling element of the CAR does not comprise a CD3^ activating element (e.g., a zlessCAR / LSR cell). It is an insight of the present disclosure that such cells (e.g., immune cells, e.g., T cells) will be dependent on a zlessCAR for tumor recognition and an Page 25 of 89 12923515v1Docket No.: 2017422-0007 LSR for T cell activation. In some embodiments, use of engineered cells (e.g., immune cells, e.g., T cells) as described herein can provide immune cell therapies, e.g., for solid tumors, that are (i) more effective, (ii) safer and (iii) more durable than certain existing cell therapies.

[0083] Among other things, the present disclosure recognizes that, in at least some embodiments, a tumor-targeting CAR may not be required to directly bind to tumor antigens; for example, in some embodiments, it can serve as an adapter CAR (or AdCAR), e.g., as described by, for example, in McCue et al., Adv Drug Deliv Rev.2022 Aug:187:114358, and Onder et al., Cancers (Basel).2023 Dec 29;16(1):168, each of which is incorporated in its entirety herein by reference. In some embodiments, tumor targeting activity of adapter CARs can be adjusted for potency and tumor antigen specificity through addition of a bifunctional biologic that recognizes both an adapter CAR and a tumor antigen, leading to bridging of CAR-expressing immune cells and tumor cells. In some embodiments, use of immune cells co-expressing LSRs with adapter CARs that comprise or lack a CD3^ domain will lead to improved efficacy and / or tolerability than is observed with existing cell therapies.

[0084] In some embodiments, a zlessCAR / LSR cell as described herein can be administered to patients with no immediate impact on tumor cells or healthy cells. In some embodiments, initial antitumor activity of an LSR can be activated by tumor lysis driven by attack by endogenous immune activity, such as TCR-mediated cytotoxicity by T cells. Alternatively or additionally, in some embodiments, anti-tumor activity of a zlessCAR / LSR cell can be activated by priming tumor cell killing using chemotherapy, monoclonal antibodies, radiation, or a short infusion of a lysis-associated antigen. Without wishing to be bound to any theory, the present disclosure describes that, once primed, zlessCAR / LSR cells would proliferate and specifically target tumor cells as lysis-associated antigens would not be released from any healthy cells that may have been lysed during a priming event. Co-stimulation

[0085] Among other things, the present disclosure recognizes that co-stimulation is a fundamental component of T cell biology and of adoptive cell therapy, specifically including CAR-T cell therapy. Effective potency of a CAR-T cell is dependent on incorporation of Page 26 of 89 12923515v1Docket No.: 2017422-0007 one or more intracellular T cell receptor co-stimulation protein elements, which serve to increase CAR-T cell persistence and enhance cell cytotoxicity in response to antigen binding to an extracellular antigen binding moiety (see, Harrison et al, 2021, the contents of which is hereby incorporated by reference herein in its entirety). First generation CARs lacked any co-stimulation elements and had limited clinical efficacy. Second generation CARs have a single co-stimulation element as exemplified by FDA approved CAR-T products Abecma (idecabtagene vicleucel), Breyanzi (lisocabtagene maraleucel), Kymriah (tisagenlecleucel) and Carvykti (ciltacabtagene autoleucel), which contain a 4-1BB co-stimulation element, and Tecartus (brexucabtagene autoleucel) and Yescarta (axicabtagene ciloleucel), which contain a CD28 co-stimulation element. Third generation CARs containing at least two co- stimulation elements have been reported to have superior antitumor activity and longer persistence (see, Harrison et al, 2021, the contents of which is hereby incorporated by reference herein in its entirety).

[0086] In some embodiments, the present disclosure utilizes a CAR comprising one or more signaling elements (e.g., T cell receptor co-stimulation elements). In some embodiments, T cell receptor co-stimulation elements are derived from CD28, 4-1BB, OX40, CD27, GITR, ICOS, as well as proprietary elements. In some embodiments, a CAR construct utilized in accordance with the present disclosure comprises a CD28 co- stimulation element. In some embodiments, a CAR utilized in accordance with the present disclosure comprises a 4-1BB co-stimulation element. In some embodiments, a CAR utilized in accordance with the present disclosure comprises a OX40 co-stimulation element. In some embodiments, a CAR utilized in accordance with the present disclosure comprises a CD27 co-stimulation element. In some embodiments, a CAR utilized in accordance with the present disclosure comprises a GITR co-stimulation element. In some embodiments, a CAR utilized in accordance with the present disclosure comprises a ICOS co-stimulation element.

[0087] In some embodiments, a CAR utilized in accordance with the present disclosure comprises one or more T cell receptor co-stimulation elements. In some embodiments, a CAR utilized in accordance with the present disclosure comprises a CD28 co-stimulation element and a 4-1BB co-stimulation element.

[0088] In some embodiments, a CAR utilized in accordance with the present disclosure comprises one or more T cell receptor co-stimulation elements as well as a CD3^ activation Page 27 of 89 12923515v1Docket No.: 2017422-0007 element. In some embodiments, a CAR utilized in accordance with the present disclosure comprises a CD28 co-stimulation element, a 4-1BB co-stimulation element, and a CD3^ activation element.

[0089] In some embodiments, a CAR utilized in accordance with the present disclosure comprises one or more T cell receptor co-stimulation elements and does not comprise a CD3^ activation element. In some embodiments, a CAR utilized in accordance with the present disclosure comprises a CD28 co-stimulation element, a 4-1BB co-stimulation element, and does not comprise a CD3^ activation element.

[0090] The present disclosure describes a LSR comprising one or more signaling elements (e.g., T cell receptor co-stimulation elements). In some embodiments, T cell receptor co-stimulation elements are derived from CD28, 4-1BB, OX40, CD27, GITR, ICOS as well as proprietary elements. In some embodiments, an exemplary LSR comprises a CD28 co-stimulation element. In some embodiments, an exemplary LSR comprises a 4- 1BB co-stimulation element. In some embodiments, an exemplary LSR comprises a OX40 co-stimulation element. In some embodiments, an exemplary LSR comprises a CD27 co- stimulation element. In some embodiments, an exemplary LSR comprises a GITR co- stimulation element. In some embodiments, an exemplary LSR comprises a ICOS co- stimulation element.

[0091] In some embodiments, a LSR as described herein comprises one or more T cell receptor co-stimulation elements. In some embodiments, an exemplary LSR comprises a CD28 co-stimulation element and a 4-1BB co-stimulation element.

[0092] In some embodiments, a LSR as described herein comprises one or more T cell receptor co-stimulation elements as well as a CD3^ activation element. In some embodiments, an exemplary LSR comprises a CD28 co-stimulation element, a 4-1BB co- stimulation element, and a CD3^ activation element. Lysis-Sensing Adaptive Cell Therapy (e.g., zlessCAR-T Therapy)

[0093] The present disclosure provides an insight that linking T cell activation to cell lysis can provide a number of therapeutic benefits. For example, the present disclosure appreciates that certain tumor environments (including specifically certain solid tumor Page 28 of 89 12923515v1Docket No.: 2017422-0007 environments) are characterized by cell lysis (e.g., have a relatively elevated level of cell lysis products). The present disclosure further provides an insight that linking of T cell activation to cell lysis can be achieved through use of a Lysis Sensor Receptor (LSR) as described and provided herein.

[0094] Among other things, the present disclosure provides preparations of cells (e.g., immune cells, e.g., T cells) engineered to express a LSR that targets an antigen made available to these receptors upon the lysis of a diseased cell (e.g., intracellular antigens / epitopes), and a CAR that targets a tumor antigen (e.g., a tumor surface antigen), wherein the activity of the CAR is attenuated via lack of a CD3^ activation element (e.g., zlessCAR / LSR cells). It is an insight of the present disclosure that such cells (e.g., immune cells, e.g., T cells) will be dependent on a zlessCAR for tumor recognition and a LSR for T cell activation. In some embodiments, zlessCAR / LSR cells will show increased tumor specificity and decreased non-specific activity.

[0095] In some embodiments, introduction of a LSR together with a CAR, wherein the CAR does not comprise a CD3^ activation element and thus is inactive on its own, into T cells can lead to activation of T cells that are in close proximity to a diseased tissue, such as a tumor. In some embodiments, such introduction achieves local activation of T cell cytotoxic activity in the vicinity of lysed cells. Lysis Sensor Receptor Constructs

[0096] Among other things, the present disclosure provides Lysis Sensor Receptor constructs (LSRs) that comprise a lysis-associated-antigen binding moiety, a transmembrane element, and a signaling element (e.g., a T cell co-stimulation element or elements).

[0097] The present disclosure describes LSR constructs that, when expressed on a cell (e.g., an immune cell, e.g., a T cell), are arranged such that their lysis-associated-antigen binding moiety is located extracellularly, e.g., such that it is arranged and constructed to bind a target lysis-associated-antigen, and a signaling element is located in cytoplasm, e.g., such that it is arranged and constructed for signal transduction and / or cell activation. Page 29 of 89 12923515v1Docket No.: 2017422-0007 Lysis-associated-antigens

[0098] The present disclosure teaches that targeting antigens associated with cell lysis (lysis-associated-antigens) as described herein can provide valuable benefits (e.g., therapeutic benefits). A lysis-associated-antigen is a protein, nucleic acid, carbohydrate, lipid or small molecule that becomes exposed at higher levels extracellularly upon lysis of a cell. Preferably, such antigens are specific to diseased cells or are present at substantially higher levels in diseased cells compared to healthy cells. In the case of tumors, such antigens include proteins encoded by tumor-specific mutations, proteins whose levels are increased due to altered signaling in tumor cells, nucleic acids expressed at higher levels in tumor cells and antigens derived from structural and metabolic changes associated with tumorigenesis. Additionally, the present disclosure describes lysis-associated-antigens such as those driven by pathogens. For example, in some embodiments, lysis-associated-antigens include genes encoded by a pathogen that are required for pathogen replication or survival within cells including components of replication and packaging machinery.

[0099] In some embodiments, a lysis-associated-antigen is an intracellular antigen that is exposed to an extracellular environment by cell lysis. As described herein, established CAR-T technologies are required to target surface antigens; established TCR technologies are required to target antigens, often intracellular antigens presented to an extracellular cell surface in MHC complexes. Lysis Sensor Receptor constructs provided by the present disclosure target intracellular antigens independent of MHC presentation. In some embodiments, a LSR described herein has a structure that resembles portions of a CAR with the exception that it targets intracellular antigens. As such, LSRs are activated only upon lysis of diseased cells and serve as a means to specifically stimulate an immune response in situations where lysis of diseased cells has occurred either as a result of disease progression, a patient’s immune response or action of another therapy.

[0100] In some embodiments, a lysis-associated-antigen may be or comprise an oncogenic protein, a protein that drives cancer cells to grow and divide uncontrollably. Exemplary tumor-specific lysis-associated-antigens include protein products of common oncogenes such as K-RAS, N-RAS, H-RAS, c-Myc, SRC family kinases and other intracellular signal transduction molecules, and intracellular elements of epidermal growth factor receptors and other receptor tyrosine kinases as well as protein products of mutated Page 30 of 89 12923515v1Docket No.: 2017422-0007 tumor suppressor proteins such as p53, BRCA1 / 2 and PTEN. The present disclosure recognizes that an important insight into tumorigenesis is the concept of oncogene addiction, which describes the concept that cancer cells become highly dependent on oncogenes for survival and thus the oncogene is likely to persist throughout the tumor and maintained as the tumor progresses. By contrast, most random mutations that occur in a tumor cell do not provide any selective advantage for tumor survival leading to tumor heterogeneity (see, Sharma and Settleman, 2007, the contents of which is hereby incorporated by reference herein in its entirety).

[0101] It is an insight of the present disclosure that mutations in RAS family members are commonly found in many cancers. KRAS mutations are found in the majority of pancreatic ductal carcinomas. In some embodiments, a LSR targets KRAS variants with a mutation occurring at amino acid 12, optionally G12D, G12V or G12C. KRAS G12D is found in roughly one third of pancreatic tumors and one sixth of colon cancers. In some embodiments a LSR targets KRAS variants with a mutation at positions other than amino acid 12. In some embodiments, a lysis-associated-antigen may be a protein product of a gene that is mutated in one percent or more of all solid tumors optionally selected from BRAF, IDH1, PI3Kalpha, P53 or others. In some embodiments, a lysis-associated-antigen may be BRAF. In some embodiments, a lysis-associated-antigen may be IDH1. In some embodiments, a lysis-associated-antigen may be PI3Kalpha. In some embodiments, a lysis- associated antigen may be P53.

[0102] In some embodiments, a lysis-associated-antigen is or comprises one or more neoepitopes. Neoepitopes are characterized as altered protein sequences that arise from mutations in tumor cells. By definition, neoepitopes are not present in healthy cells and are tumor-specific. Whereas there is a potential for neoepitopes to be recognized by TCRs, neoepitopes that can be recognized in this manner are limited to those than can be presented by MHC class II receptors in an individual patient. It is an insight of the present disclosure that there is no such limitation on neoepitopes that could be targeted by LSRs, with the only requirement that they be present upon cell lysis. Page 31 of 89 12923515v1Docket No.: 2017422-0007 Lysis-associated-antigen binding moiety

[0103] Among other things, the present disclosure provides technologies (e.g., compositions, methods, producer cells, etc.) that are or comprise LSR constructs described herein. In some embodiments, technologies described herein comprise a LSR construct comprising a lysis-associated-antigen binding moiety. In some embodiments, a lysis- associated-antigen binding moiety binds specifically to a lysis-associated-antigen, as described herein.

[0104] In some embodiments, a lysis-associated-antigen binding moiety is or comprises antigen binding elements of an immunoglobulin (e.g., an antibody, a T cell receptor, etc.). In some embodiments, a lysis-associated-antigen binding moiety is or comprises a set of CDRs.

[0105] In some embodiments, a lysis-associated-antigen binding moiety is or comprises a single chain variable fragment (scFv).

[0106] It is an insight of the present disclosure that antibodies and scFvs have been described for potential lysis-associated-antigens demonstrating that these antigens are readily recognizable by antibody-based proteins generated by various technologies. The findings of naturally occurring antibodies to these antigens in cancer patients is consistent with extracellular exposure of these antigens to components of the immune system (see, for example, Collins & Pasca Di Magliano 2014, the contents of which is hereby incorporated by reference herein in its entirety). Thus, in some embodiments, lysis of tumor cells will make such antigens available for binding to LSRs.

[0107] In some embodiments, a lysis-associated-antigen binding moiety comprises a KRAS binding moiety as described by Singh et al., 2022, the contents of which is hereby incorporated by reference herein in its entirety. In some embodiments, a lysis-associated- antigen binding moiety comprises a KRAS binding moiety as described by Shin et al., 2017, the contents of which is hereby incorporated by reference herein in its entirety. In some embodiments, a lysis-associated antigen-binding moiety comprises a KRAS binding moiety as described by U.S. Pat. No.11,174,314 B2, the relevant disclosures of which are incorporated by reference herein. In some embodiments, a lysis-associated-antigen binding moiety comprises a STAT3 binding moiety as described by Singh et al., 2022. Page 32 of 89 12923515v1Docket No.: 2017422-0007

[0108] Among other things, in some embodiments, an exemplary LSR construct comprises a KRAS mutant-specific scFV sequence according to SEQ ID NO: 1.

[0109] Exemplary KRAS mutant-specific scFV sequence (SEQ ID NO: 1) EVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMHWVRQAPGKGLEWVAVISHDG SKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDTAVYYCATSLYSSMDLWGQGTT VTVSSGSTSGSGKPGSGEGSTKGQSVVTQPPSVSAAPGQKVTISCSGSNSNIGKNYV SWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGTSASLAISGLQSEDEADYYCAAW DDKFGVHWVFGGGTKLTVL

[0110] Critical to immune cell activation is the ability of binding of a ligand to an extracellular element to trigger a change in intracellular signaling. For many immune cell receptors, activation of cell signaling is mediated by protein-protein interactions, driven by receptor multimerization. For traditional CARs, this multimerization can be a result of CAR binding to multiple copies of an antigen on a cell surface of a target cell. Among other things, the present disclosure describes that LSR multimerization can be a result of antigens bound to intracellular cell structures such as membranes, protein complexes, structural elements, nucleic acids and glycoproteins. Alternatively or additionally, antigens released upon cell lysis may bind to extracellular elements resulting in multimer structures.

[0111] It is an insight of the present disclosure that a LSR could target more than one antigen as exemplified by a bispecific CAR that targets both CS1 and BCMA (see, for example, Zha et al., 2020, the contents of which is hereby incorporated by reference herein in its entirety). In some embodiments, a LSR cell therapy may comprise immune cells containing LSRs targeting different antigens or different antigenic sites on a single antigen (see, Han et al., 2019, the contents of which is hereby incorporated by reference herein in its entirety). For example, in some embodiments LSRs that target N-terminal domains and C- terminal domains of an antigen, such as P53 are introduced into an immune cell. Thus, in some embodiments, multimerization of distinct LSRs by an antigen, even in a monomeric form, will trigger immune cell activation. Chimeric Antigen Receptor Constructs

[0112] Among other things, the present disclosure utilizes Chimeric Antigen Receptor constructs (CARs) that comprise an antigen-binding moiety, a transmembrane element, and a signaling element (e.g., a T cell co-stimulation element or elements). In some Page 33 of 89 12923515v1Docket No.: 2017422-0007 embodiments, the present disclosure utilizes CAR constructs that, when expressed on a cell (e.g., an immune cell, e.g., a T cell), are arranged such that their antigen-binding moiety is located extracellularly, e.g., such that it is arranged and constructed to bind a target antigen, and their signaling element is located in cytoplasm, e.g., such that it is arranged and constructed for signal transduction and / or cell activation.

[0113] In some embodiments, the present disclosure utilizes adapter CAR constructs that comprise an adapter-specific recognition domain, a transmembrane element, and a signaling element (e.g., a T cell co-stimulation element or elements) as described by, for example, Seitz et al., 2021, the contents of which is hereby incorporated by reference herein in its entirety. An adapter molecule comprising an antigen-binding moiety and a CAR-binding moiety can reversibly redirect immune cells comprising such adapter CARs to a tumor- associated antigen or to different tumor-associated antigens. Moreover, activity of an adapter CAR is dependent on binding with an adapter molecule. Exemplary antigens

[0114] The present disclosure teaches immune cells comprising a CAR that targets an antigen (e.g., a tumor antigen, e.g., a tumor surface antigen). In some embodiments, exemplary antigens targeted by a CAR utilized herein may be or comprise a nucleic acid, carbohydrate, lipid, or small molecule that becomes exposed at higher levels extracellularly upon lysis of a cell. Preferably, such antigens are specific to diseased cells or are present at substantially higher levels in diseased cells compared to healthy cells. In the case of tumors, such antigens include proteins encoded by tumor-specific mutations, proteins whose levels are increased due to altered signaling in tumor cells, nucleic acids expressed at higher levels in tumor cells and antigens derived from structural and metabolic changes associated with tumorigenesis. Additionally, the present disclosure describes antigens such as those driven by pathogens. For example, in some embodiments, antigens include genes encoded by a pathogen that are required for pathogen replication or survival within cells including components of replication and packaging machinery.

[0115] In some embodiments, an antigen is a tumor surface antigen.

[0116] In some embodiments, an antigen is a surface-expressed antigen. Page 34 of 89 12923515v1Docket No.: 2017422-0007

[0117] In some embodiments, an antigen may be or comprise an oncogenic protein, a protein that drives cancer cells to grow and divide uncontrollably.

[0118] In some embodiments, an antigen is B7-H3. In some embodiments, an antigen is CD171. In some embodiments, an antigen is CEA. In some embodiments, an antigen is CLD18.2. In some embodiments, an antigen is EGFR. In some embodiments, an antigen is EpCAM. In some embodiments, an antigen is GD2. In some embodiments, an antigen is GPC3. In some embodiments, an antigen is HER2. In some embodiments, an antigen is SSTR2. In some embodiments, an antigen is IL12Ra2. In some embodiments, an antigen is MAGE-A3. In some embodiments, an antigen is mesothelin. In some embodiments, an antigen is Mucin-1. In some embodiments, an antigen is NKG2D. In some embodiments, an antigen is PSMA. In some embodiments, an antigen is ROR1. In some embodiments, an antigen is ZAP70.

[0119] Among other things, the present disclosure provides CAR constructs comprising an antigen binding moiety that targets mesothelin. Mesothelin overexpression has been documented in a variety of solid tumors. Moreover, CAR T-cells targeting mesothelin have been clinically investigated in mesothelioma, epithelial ovarian cancer, pancreatic ductal adenocarcinoma (PDAC), lung cancers, uterine cancers, triple-negative breast cancer (TNBC), gastric cancer (GC), CRC, esophagus cancer, hepatocellular carcinoma (HCC), as well as neuroendocrine tumors / Merkel cell carcinoma (see, for example, Sorkhabi et al., Front Immunol.2023; 14: 1113882, the contents of which is hereby incorporated by reference herein in its entirety). Antigen-binding moiety

[0120] Among other things, the present disclosure provides technologies (e.g., compositions, methods, producer cells, etc.) that are or comprise CAR constructs described herein. In some embodiments, technologies described herein comprise a CAR construct comprising an antigen-binding moiety. In some embodiments, an antigen-binding moiety binds specifically to a tumor antigen, as described herein.

[0121] In some embodiments, an antigen-binding moiety is or comprises antigen binding elements of an immunoglobulin (e.g., an antibody, a T cell receptor, etc.). In some embodiments, an antigen-binding moiety is or comprises a set of CDRs. Page 35 of 89 12923515v1Docket No.: 2017422-0007

[0122] In some embodiments, an antigen-binding moiety is or comprises a single chain variable fragment (scFv).

[0123] It is an insight of the present disclosure that antibodies and scFvs have been described for potential tumor antigens demonstrating that these antigens are readily recognizable by antibody-based proteins generated by various technologies.

[0124] In some embodiments, a CAR utilized in accordance with the present disclosure comprises an anti-mesothelin antigen-binding moiety. In some embodiments, a CAR utilized in accordance with the present disclosure comprises an anti-HER2 antigen-binding moiety. In some embodiments, a CAR utilized in accordance with the present disclosure comprises an anti-EGFR antigen-binding moiety. In some embodiments, a CAR utilized in accordance with the present disclosure comprises an anti-PSMA antigen-binding moiety. In some embodiments, a CAR utilized in accordance with the present disclosure comprises an anti-SSTR2 antigen-binding moiety. In some embodiments, a CAR utilized in accordance with the present disclosure comprises an anti-EpCAM antigen-binding moiety. In some embodiments, a CAR utilized in accordance with the present disclosure comprises an anti- GD2 antigen-binding moiety. Transmembrane element

[0125] In many embodiments, a Lysis Sensor Receptor construct and / or a Chimeric Antigen Receptor (CAR) construct for use in accordance with the present disclosure comprises a transmembrane element that, for example, may link a lysis-associated-antigen binding moiety as described herein to a signaling element as described herein. Those skilled in the art will be familiar with a variety of transmembrane elements that can be used in the practice of the present disclosure. Alternatively or additionally, in some embodiments, a transmembrane element of a LSR and / or CAR construct regulates LSR and / or CAR expression and / or signaling activity (see, for example, Fujiwara et al., 2020, the contents of which is hereby incorporated by reference herein in its entirety).

[0126] In some embodiments, a transmembrane element may correspond to one that is found in a naturally occurring protein. Alternatively or additionally, in some embodiments, a transmembrane element may be designed or otherwise engineered, for example, to be or include a substantially hydrophobic segment that is thermodynamically stable in a cell Page 36 of 89 12923515v1Docket No.: 2017422-0007 membrane, whether or not its sequence is found in any known transmembrane protein (e.g., any known human transmembrane protein). Examples of synthetic transmembrane domains that are known in the art include, for example, those described in U.S. Pat. No.7,052,906 Bl and PCT Publication No. WO 2000 / 032776 A2, the relevant disclosures of which are incorporated by reference herein.

[0127] In some embodiments, a transmembrane element for use in accordance with the present disclosure corresponds to one found in a membrane protein selected from the group consisting of: CD8^, CDS~, 4-1BB / CD137, CD28, CD34, CD4, FcERiy, CD16, OX40 / CD134, CD3s, CD3E, CD3y, CD3o, TCRa, CD32, CD64, VEGFR2, FAS, and FGFR2B. In some embodiments, a transmembrane element for use in accordance with the present disclosure corresponds to one found in CD8a. In some examples, a transmembrane element for use in accordance with the present disclosure corresponds to one found in 4- lBB / CD137. In some embodiments, a transmembrane element for use in accordance with the present disclosure corresponds to one found in CD28 or CD34. In some embodiments, a transmembrane element for use in accordance with the present disclosure corresponds to one found in CD8a. Signaling element

[0128] As described herein, effective CAR-T cell therapy requires co-stimulation to increase CAR-T cell persistence and enhance cell cytotoxicity in response to antigen binding. In many embodiments, a LSR utilized in accordance with the present disclosure comprises one or more signaling elements (e.g., co-stimulation element(s)) which leads to immune effector function as described herein. In many embodiments, a CAR utilized in accordance with the present disclosure comprises one or more signaling elements (e.g., co- stimulation element(s)) which leads to immune effector function as described herein. Those skilled in the art, reading the present disclosure, would be aware of appropriate source(s) of co-stimulation elements for particular applications as described herein.

[0129] Reports have described successful Co-CAR constructs that utilize, for example, co-stimulation elements from CD28 and OX40 proteins (see, Omer et al., Cancer Immunol Res.2022 Apr 1;10(4):512-524.). Exemplary signaling elements (e.g., co-stimulation element(s)) that may be used in accordance with the present disclosure include those Page 37 of 89 12923515v1Docket No.: 2017422-0007 selected from the group consisting of MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and a ligand that specifically binds with CD83. Exemplary co- stimulation elements for use in accordance with the present disclosure are described in U.S. Pat. No.11,084,880 B2, incorporated herein by reference. In some embodiments, a co- stimulation element is derived from a group consisting of CD28, OX40, 4I-BB, GITR, ICOS-1, CD27, AP10, and any combination thereof. In some embodiments, a co- stimulation element comprises a CD3^ (CD3^ or CD3C) activating element. In some embodiments, a co-stimulation element does not comprise a CD3^ (CD3^ or CD3C) activating element.

[0130] In some embodiments, a LSR construct may comprise more than one co- stimulation element (e.g., 2, 3, or more). In some embodiments, a LSR construct comprises two or more of the same co-stimulation elements. In some embodiments, a LSR construct comprises two or more co-stimulation elements from different co- stimulatory proteins, such as any two or more co-stimulatory proteins described herein.

[0131] The present disclosure describes LSR constructs comprising one or more signaling elements (e.g., T cell receptor co-stimulation elements). In some embodiments, an exemplary LSR construct comprises one or more T cell receptor co-stimulation elements. In some embodiments, T cell receptor co-stimulation elements are derived from CD28, 4-1BB, OX40, CD27, GITR, ICOS as well as proprietary elements. In some embodiments, an Page 38 of 89 12923515v1Docket No.: 2017422-0007 exemplary LSR construct comprises a CD28 co-stimulation element. An exemplary CD28 co-stimulation element sequence may be or comprise a sequence according to SEQ ID NO: 2. In some embodiments, an exemplary LSR construct comprises a 4-1BB co-stimulation element. An exemplary 4-1BB co-stimulation element sequence may be or comprise a sequence according to SEQ ID NO: 3. In some embodiments, an exemplary LSR construct comprises a OX40 co-stimulation element. In some embodiments, an exemplary LSR construct comprises a CD27 co-stimulation element. In some embodiments, an exemplary LSR construct comprises a GITR co-stimulation element. In some embodiments, an exemplary LSR construct comprises a ICOS co-stimulation element.

[0132] In some embodiments, an exemplary LSR construct comprises a CD28 co- stimulation element and a 4-1BB co-stimulation element.

[0133] In some embodiments, an exemplary LSR construct comprises one or more T cell receptor co-stimulation elements as well as a CD3^ activation element. An exemplary CD3^ activation element sequence may be or comprise a sequence according to SEQ ID NO: 4. In some embodiments, an exemplary LSR construct does not comprise a CD3^ activation element.

[0134] In some embodiments, an exemplary LSR comprises a CD28 co-stimulation element, a 4-1BB co-stimulation element, and a CD3^ activation element.

[0135] In some embodiments, a CAR construct utilized in accordance with the present disclosure may comprise more than one co-stimulation element (e.g., 2, 3 or more). In some embodiments, a CAR construct utilized in accordance with the present disclosure comprises two or more of the same co-stimulation elements. In some embodiments, a CAR construct utilized in accordance with the present disclosure comprises two or more co-stimulation elements from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein.

[0136] The present disclosure utilizes CAR constructs comprising one or more signaling elements (e.g., T cell receptor co-stimulation elements). In some embodiments, a CAR construct utilized in accordance with the present disclosure comprises one or more T cell receptor co-stimulation elements. In some embodiments, T cell receptor co-stimulation elements are derived from CD28, 4-1BB, OX40, CD27, GITR, ICOS as well as proprietary Page 39 of 89 12923515v1Docket No.: 2017422-0007 elements. In some embodiments, a CAR construct utilized in accordance with the present disclosure comprises a CD28 co-stimulation element. An exemplary CD28 co-stimulation element sequence may be or comprise a sequence according to SEQ ID NO: 2. In some embodiments, a CAR construct utilized in accordance with the present disclosure comprises a 4-1BB co-stimulation element. An exemplary 4-1BB co-stimulation element sequence may be or comprise a sequence according to SEQ ID NO: 3. In some embodiments, a CAR construct utilized in accordance with the present disclosure comprises a OX40 co- stimulation element. In some embodiments, an exemplary CAR construct comprises a CD27 co-stimulation element. In some embodiments, a CAR construct utilized in accordance with the present disclosure comprises a GITR co-stimulation element. In some embodiments, a CAR construct utilized in accordance with the present disclosure comprises a ICOS co-stimulation element.

[0137] In some embodiments, a CAR construct utilized in accordance with the present disclosure comprises a CD28 co-stimulation element and a 4-1BB co-stimulation element.

[0138] In some embodiments, a CAR construct utilized in accordance with the present disclosure does not comprise a CD3^ activation element (e.g., zlessCAR).

[0139] In some embodiments, a CAR utilized in accordance with the present disclosure a CD28 co-stimulation element, a 4-1BB co-stimulation element, and does not comprise a CD3^ activation element.

[0140] Exemplary CD28 co-stimulation element sequence (SEQ ID NO: 2) ASAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRLGPTRKHYQPYAPPRDFAAYRS

[0141] Exemplary 4-1BB co-stimulation element sequence (SEQ ID NO: 3) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE

[0142] Exemplary CD3^ activation element sequence (SEQ ID NO: 4) LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR Page 40 of 89 12923515v1Docket No.: 2017422-0007 Other element(s)

[0143] Among other things, the present disclosure describes a LSR construct comprising one or more elements that regulate activity of a LSR. Among other things, the present disclosure describes a CAR construct comprising one or more elements that regulate activity of a CAR. For example, in some embodiments, a LSR construct and / or a CAR construct may comprise a masking element that is cleaved off or moved in a tumor microenvironment. In some embodiments, constructs of the present disclosure may comprise a masking element as described by U.S. Pat. No.9,120,853 B2, the contents of which are hereby incorporated by reference in its entirety.

[0144] The potency and survival of zlessCAR / LSR cell therapies is dependent in part to the strength of a co-stimulation signal transmitted by a LSR receptor. It is an insight of the present disclosure that four factors may be optimized to generate an effective therapy: (1) the strength and number of co-stimulation elements, (2) the expression density of a LSR on engineered immune cells, (3) the affinity of a lysis-antigen-binding moiety for a target antigen, and (4) the density and availability of an antigen after cell lysis (see, Rodriguez- Marquez et al., 2022, Majzner et al., 2020, the contents of which is hereby incorporated by reference herein in its entirety).

[0145] Among other things, in some embodiments, a LSR construct and / or a CAR construct as described herein comprises a signaling peptide sequence. An exemplary signaling peptide sequence may be or comprise a sequence according to SEQ ID NO: 5.

[0146] Exemplary signaling peptide sequence (SEQ ID NO: 5) MLLLVTSLLLCELPHPAFLLIP

[0147] Among other things, in some embodiments, a LSR construct and / or a CAR construct as described herein further comprises, a polypeptide tag (e.g., a FLAG-tag, or FLAG octapeptide, or FLAG epitope). In some embodiments, a polypeptide tag (e.g., a FLAG-tag, or FLAG octapeptide, or FLAG epitope) is used to assess expression of a LSR and / or a CAR in T cells. An exemplary polypeptide tag sequence may be or comprise a sequence according to SEQ ID NO: 6. Page 41 of 89 12923515v1Docket No.: 2017422-0007

[0148] Exemplary polypeptide tag sequence (SEQ ID NO: 6) DYKDDDDK

[0149] Those of skill in the art, reading the present disclosure, would be aware of polypeptide tags and methods of adding a polypeptide tag using recombinant DNA technology (see, e.g., WO 2017 / 172952 A1, the contents of which is hereby incorporated by reference herein in its entirety. Exemplary Lysis Sensor Receptor Construct Sequences

[0150] Among other things, in some embodiments, the present disclosure provides technologies (e.g., compositions, vectors, virions, producer cells). In some embodiments, such technologies comprise a single construct. In some embodiments, such technologies comprise multiple constructs. In some embodiments, the present disclosure provides compositions or preparation of virions each comprised of a single construct as described herein. In some embodiments, a single construct may deliver a polynucleotide that encodes an exemplary LSR. In some embodiments, a construct is or comprises a LSR construct. In some embodiments, a single construct may deliver both an exemplary CAR (e.g., zlessCAR) and an exemplary LSR.

[0151] In some embodiments, a single construct composition or system may comprise any or all of the exemplary construct components described herein. In some embodiments, an exemplary single construct is at least 85%, 90%, 95%, 98% or 99% identical to the sequences described herein. One skilled in the art would recognize that constructs may undergo additional modifications including codon-optimization, introduction of novel but functionally equivalent (e.g., silent mutations), addition of reporter sequences, and / or other routine modification.

[0152] In some embodiments, constructs, virions, populations of virions, or producer cells comprise a LSR polypeptide sequence that shows at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% overall sequence identity with any one of SEQ ID NOs: 7, 9, 11, or 12. In some embodiments, constructs, virions, populations of virions, or producer cells comprise a LSR polynucleotide sequence that shows at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at Page 42 of 89 12923515v1Docket No.: 2017422-0007 least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% overall sequence identity with any one of SEQ ID NOs: 8 or 10.

[0153] Exemplary KRAS-LSR construct 1 polypeptide sequence (SEQ ID NO: 7) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMH WVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDT AVYYCATSLYSSMDLWGQGTTVTVSSGSTSGSGKPGSGEGSTKGQSVVTQPPSVSA APGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGT SASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTVLDYKDDDDKASAIEV MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWVRSKRSRLLHSDYMNMTPRRLGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0154] Exemplary KRAS-LSR construct 1 polynucleotide sequence (SEQ ID NO: 8) ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCT CCTGATCCCAGAAGTGCAACTTGTACAGTCTGGAGGTGGCGTTGTACAGCCTGG GCGGAGCCTCCGACTCAGCTGCGCCGCATCAGGATTTACAAGCCGACATCCGGG AATGCACTGGGTGCGCCAAGCACCAGGGAAAGGCTTGGAGTGGGTGGCCGTTAT TAGCCACGACGGTTCTAAAAAGTACTATGCAGACTCCGTGAAGGGGCGGTTTACC ATCTCCAGGGATAACTCTAAGAACACGCTGTTTGTCCAACTGTCTTCCTTGCGGC CGGAAGATACTGCGGTCTACTACTGCGCTACTTCTCTTTACAGCAGCATGGACCT GTGGGGCCAAGGAACCACCGTGACTGTGAGTAGCGGCTCCACCTCTGGATCCGG CAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCCAGTCCGTAGTTACCCAGCC TCCTAGTGTAAGCGCGGCCCCGGGTCAAAAAGTGACGATCAGCTGTTCTGGCTCT AACAGTAACATTGGGAAGAATTATGTAAGTTGGTTTCAACAGGTGCCGGGCACG GCACCCAAGCTCCTTATTTTTGAGGATAACCAACGACCATCAGGTATCCCCGACA GATTTTCTGCGTCTAAATCTGGTACGTCCGCGAGCCTTGCTATAAGTGGTCTTCAG TCAGAGGATGAAGCTGACTACTATTGTGCTGCCTGGGATGATAAATTTGGCGTCC ATTGGGTCTTTGGTGGGGGGACAAAGCTCACTGTATTGGACTACAAAGACGATG ACGACAAGGCTAGCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGA GAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCC CCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTC CTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCAAGACGCCT AGGTCCCACTCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCC TATCGCTCCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTAT GAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA AGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACG CTCCAGCTTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG GGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG Page 43 of 89 12923515v1Docket No.: 2017422-0007 AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGAC ACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

[0155] Exemplary zlessKRAS-LSR construct 2 polypeptide sequence (SEQ ID NO: 9) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMH WVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDT AVYYCATSLYSSMDLWGQGTTVTVSSGSTSGSGKPGSGEGSTKGQSVVTQPPSVSA APGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGT SASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTVLDYKDDDDKASAIEV MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWVRSKRSRLLHSDYMNMTPRRLGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0156] Exemplary zlessKRAS-LSR construct 2 polynucleotide sequence (SEQ ID NO: 10) ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCT CCTGATCCCAGAAGTGCAACTTGTACAGTCTGGAGGTGGCGTTGTACAGCCTGG GCGGAGCCTCCGACTCAGCTGCGCCGCATCAGGATTTACAAGCCGACATCCGGG AATGCACTGGGTGCGCCAAGCACCAGGGAAAGGCTTGGAGTGGGTGGCCGTTAT TAGCCACGACGGTTCTAAAAAGTACTATGCAGACTCCGTGAAGGGGCGGTTTACC ATCTCCAGGGATAACTCTAAGAACACGCTGTTTGTCCAACTGTCTTCCTTGCGGC CGGAAGATACTGCGGTCTACTACTGCGCTACTTCTCTTTACAGCAGCATGGACCT GTGGGGCCAAGGAACCACCGTGACTGTGAGTAGCGGCTCCACCTCTGGATCCGG CAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCCAGTCCGTAGTTACCCAGCC TCCTAGTGTAAGCGCGGCCCCGGGTCAAAAAGTGACGATCAGCTGTTCTGGCTCT AACAGTAACATTGGGAAGAATTATGTAAGTTGGTTTCAACAGGTGCCGGGCACG GCACCCAAGCTCCTTATTTTTGAGGATAACCAACGACCATCAGGTATCCCCGACA GATTTTCTGCGTCTAAATCTGGTACGTCCGCGAGCCTTGCTATAAGTGGTCTTCAG TCAGAGGATGAAGCTGACTACTATTGTGCTGCCTGGGATGATAAATTTGGCGTCC ATTGGGTCTTTGGTGGGGGGACAAAGCTCACTGTATTGGACTACAAAGACGATG ACGACAAGGCTAGCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGA GAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCC CCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGGGGAGTC CTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCAAGACGCCT AGGTCCCACTCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCC TATCGCTCCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTAT GAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA AGAAGAAGAAGGAGGATGTGAACTG Page 44 of 89 12923515v1Docket No.: 2017422-0007

[0157] Exemplary zlessMeso-CAR / KRAS-LSR dual expression polypeptide sequence (SEQ ID NO: 11) MALPVTALLLPLALLLHAARPASQVQLVQSGAEVEKPGASVKVSCKASGYTFTDYY MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRS DDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSLSA SVGDRVTITCRASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCLQTYTTPDFGPGTKVEIKLEKPTTTPAPRPPTPAPTIASQPLSL RPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRAKRSGSGATNFSLLKQAG DVEENPGPMLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGVVQPGRSLRLSCAASGFT SRHPGMHWVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLFVQLS SLRPEDTAVYYCATSLYSSMDLWGQGTTVTVSSGSTSGSGKPGSGEGSTKGQSVVTQ PPSVSAAPGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIPDRFS ASKSGTSASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTVLDYKDDDD KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0158] Exemplary Meso-CAR / KRAS-LSR dual expression polypeptide sequence (SEQ ID NO: 12) MALPVTALLLPLALLLHAARPASQVQLVQSGAEVEKPGASVKVSCKASGYTFTDYY MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRS DDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSLSA SVGDRVTITCRASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCLQTYTTPDFGPGTKVEIKLEKPTTTPAPRPPTPAPTIASQPLSL RPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRSGSGATNFSLLK QAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGVVQPGRSLRLSCAA SGFTSRHPGMHWVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLF VQLSSLRPEDTAVYYCATSLYSSMDLWGQGTTVTVSSGSTSGSGKPGSGEGSTKGQS VVTQPPSVSAAPGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIP DRFSASKSGTSASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTVLDYKD DDDKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Page 45 of 89 12923515v1Docket No.: 2017422-0007 Exemplary Chimeric Antigen Receptor Construct Sequences

[0159] Among other things, the present disclosure provides Chimeric Antigen Receptor constructs (CARs) that comprise an antigen-binding moiety, a transmembrane element, and a signaling element (e.g., a T cell co-stimulation element or elements).

[0160] In some embodiments, an antigen-binding moiety binds to a tumor-specific antigen. In some embodiments, an antigen-binding moiety binds to a tumor-surface expressed protein.

[0161] In some embodiments, an antigen-binding moiety comprises an anti-mesothelin binding moiety. Those skilled in the art would be aware of tumor-specific antigens which can be bound by an antigen-binding moiety in the practice of the present disclosure.

[0162] In some embodiments, constructs, virions, populations of virions, or producer cells described herein comprise a CAR polypeptide sequence that shows at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% overall sequence identity with that of SEQ ID NO: 13. In some embodiments, constructs, virions, populations of virions, or producer cells described herein comprise a CAR polynucleotide sequence that shows at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% overall sequence identity with that of SEQ ID NO: 14.

[0163] Exemplary CAR construct 1 polypeptide sequence (SEQ ID NO: 13) MALPVTALLLPLALLLHAARPASQVQLVQSGAEVEKPGASVKVSCKASGYTFTDYY MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRS DDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSLSA SVGDRVTITCRASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCLQTYTTPDFGPGTKVEIKLEKPTTTPAPRPPTPAPTIASQPLSL RPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0164] Exemplary CAR construct 1 polynucleotide sequence (SEQ ID NO: 14) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCG CCAGGCCGGCTAGCCAGGTTCAACTCGTTCAAAGTGGAGCAGAAGTGGAGAAG CCAGGAGCCTCCGTGAAAGTATCCTGCAAAGCTTCAGGTTATACATTCACGGACT ATTACATGCATTGGGTGCGCCAAGCACCCGGACAAGGCCTTGAATGGATGGGCTG Page 46 of 89 12923515v1Docket No.: 2017422-0007 GATCAATCCGAACTCAGGCGGTACAAATTATGCCCAAAAGTTTCAAGGCCGCGTT ACGATGACCCGGGACACATCAATCTCTACAGCATATATGGAACTGAGTCGACTTA GAAGCGACGACACAGCAGTGTACTATTGCGCCAGCGGGTGGGACTTTGACTATT GGGGGCAGGGAACATTGGTTACAGTTAGTAGCGGCGGGGGAGGGAGTGGCGGG GGAGGATCAGGAGGAGGCGGTTCCGATATTGTAATGACTCAGTCTCCCTCCTCCT TGTCAGCATCTGTAGGCGATAGAGTGACAATAACGTGCCGGGCATCTCAGAGTAT CAGATACTACTTGAGCTGGTACCAGCAAAAGCCCGGGAAGGCCCCTAAGCTCCT GATATATACTGCTTCCATCTTGCAGAATGGCGTCCCAAGCCGATTTAGTGGCAGCG GAAGCGGAACTGATTTTACATTGACCATTTCTAGTCTTCAGCCCGAGGACTTCGC AACTTATTACTGCTTGCAAACCTACACCACACCCGACTTCGGTCCCGGCACCAAA GTCGAAATAAAACTCGAGAAGCCCACCACGACGCCAGCGCCGCGACCACCAAC ACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCG GCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATA AGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCT AGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTG TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATG GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGTGA Encoding Nucleic Acids

[0165] In some embodiments, the present disclosure provides nucleic acids encoding LSRs as described herein, and compositions that comprise and / or deliver them (e.g., to cells as described herein). In some embodiments, the present disclosure provides nucleic acids encoding CARs as described herein, and compositions that comprise and / or deliver them (e.g., to cells as described herein).

[0166] Among other things, the present disclosure provides that some LSR constructs as described herein are polynucleotide constructs. In some embodiments, the present disclosure provides that some CAR constructs as described herein are polynucleotide constructs. In some embodiments, a polynucleotide construct may comprise a nucleic acid encoding a LSR construct and a nucleic acid encoding a CAR construct. Polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and constructs that incorporate a polynucleotide comprising a coding sequence operably linked to an expression control sequence, wherein the coding sequence encodes a LSR. Those of skill in the art will be capable of selecting suitable constructs, as well as cells, for making any of a nucleic acids described herein. In some embodiments, a construct is a plasmid (i.e., a circular DNA molecule that can autonomously replicate inside a cell). In some embodiments, a construct can be a cosmid (e.g., pWE or sCos series). Page 47 of 89 12923515v1Docket No.: 2017422-0007

[0167] Those of skill in the art, reading the present disclosure, would be aware of methods of generating a polynucleotide construct in accordance with the present disclosure. Examples of methods of generating a polynucleotide construct as described herein (e.g., in vitro transcription, enzymatic ligation, genetic engineering techniques), as described by, for example, Wood, 1982, incorporated in its entirety herein by reference.

[0168] Those of skill in the art, reading the present disclosure, would be aware of methods for delivery of nucleic acids (e.g., to cells as described herein). Examples of methods for delivery of a polynucleotide construct (e.g., a LSR construct, e.g., a CAR construct) that are known in the art include, for example, those described in U.S. Pat. No. 10,898,574 B2, Hou et al., 2021, and U.S. Pat. No.11,084,880 B2, each of which is incorporated in its entirety herein by reference.

[0169] Among other things, the present disclosure describes a vector comprising a polynucleotide construct encoding a LSR to facilitate introduction into immune cells. In some embodiments, the present disclosure describes a vector comprising a polynucleotide construct encoding a CAR to facilitate introduction into immune cells. In some embodiments, a viral vector can be a retrovirus vector (including an oncoretrovirus vector, a lentivirus vector, and a pseudo type vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a sendai virus vector, an Epstein-Barr virus (EBV) vector, a HSV vector among others. Examples of lentiviral vectors for use in accordance with the present disclosure include, for example, human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2); visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). Examples of viral vectors that may be used in accordance with the present disclosure are described in U.S. Pat. No.11,382,965 B2, the contents of which are incorporated in its entirety herein by reference.

[0170] In some embodiments, a viral vector lacking replicating ability so as not to self replicate in an infected cell is preferably used.

[0171] Among other things, in some embodiments, the present disclosure provides producer cells so that virions are produced in the producer cell that comprise the viral capsid Page 48 of 89 12923515v1Docket No.: 2017422-0007 comprising a polynucleotide construct as described herein. Methods of introducing and expressing genes in a cell are known in the art. In some embodiments, a polynucleotide construct encoding a LSR construct described herein is transferred into a producer cell. In some embodiments, a polynucleotide construct encoding a CAR construct described herein is transferred into a producer cell.

[0172] Moreover, the present disclosure describes a preparation of virions comprising a viral capsid, a LSR construct, and a CAR construct utilized in accordance with the present disclosure to facilitate introduction into cells (e.g., immune cells).

[0173] Among other things, in some embodiments, a polynucleotide construct encoding a LSR construct described herein is operably linked to a promoter and incorporated into an expression vector. In some embodiments, the expression vector may be provided to a cell in the form of a viral vector. In some embodiments, a polynucleotide construct encoding a CAR construct utilized in accordance with the present disclosure is operably linked to a promoter and incorporated into an expression vector. In some embodiments, the expression vector may be provided to a cell in the form of a viral vector.

[0174] Those of skill in the art, reading the present disclosure, would be aware of methods of producing engineered immune cells including, for example, the use of viral vectors, transposons, electroporation, and increasing cell membrane permeability. In some embodiments, a LSR coding sequence and a zlessCAR coding sequence may be introduced into cells (e.g., immune cells)simultaneously or sequentially using two independent constructs. In some embodiments, a LSR coding sequence and a zlessCAR coding sequence may be introduced into cells (e.g., immune cells) as a single construct. Such a single construct can drive independent expression of both a LSR and zlessCAR or can express them in tandem separated by a potential protease cleavage site (see, for example, He et al., Blood.2020 Mar 5;135(10):713-723., the contents of which are hereby incorporated by reference in its entirety). In some embodiments, cells (e.g., immune cells) engineered to express a zlessCAR and a LSR may be generated in vivo (see, for example, Short et al. Trends Pharmacol Sci.2024 May;45(5):406-418, the contents of which are hereby incorporated by reference in its entirety). Page 49 of 89 12923515v1Docket No.: 2017422-0007 LSR and CAR Activation

[0175] The present disclosure contemplates a variety of mechanisms that may be involved in LSR activation upon antigen (i.e., lysis-associated-antigen) binding and in CAR activation upon antigen binding.

[0176] For example, in some embodiments, antigen binding may induce a conformational change, for example in an intracellular domain of the LSR and / or CAR, thereby achieving direct activation. Alternatively or additionally, in some embodiments, antigen binding may alter interaction(s) between a LSR and / or a CAR and one or more other agents (e.g., other proteins), thereby achieving transduction of a relevant signal. Still further, alternatively or additionally, in some embodiments, antigen binding could trigger dimerization or higher order multimerization of a LSR and / or a CAR (e.g., through binding of dimeric or oligomeric antigen ligands and / or through binding of multiple ligands that form multimeric structures, for example). Yet further alternatively or additionally, in some embodiments, different LSRs may target different antigenic regions (e.g., epitopes) of a same target antigen (e.g., target protein, carbohydrate, complex, etc.), such that antigen binding by two or more such LSRs co-localizes different LSRs, thereby achieving signal activation. In some embodiments, different CARs may target different antigenic regions (e.g., epitopes) of a same target antigen (e.g., target protein, carbohydrate, complex, etc.), such that antigen binding by two or more such CARs co-localizes different CARs, thereby achieving signal activation.

[0177] Those skilled in the art will be aware of different elements, or combinations thereof that may be included in LSRs and / or CARs provided herein to accomplish signal transduction by one or more such mechanisms (see, for example, Schlessinger, 1998, and Wu, 2013, each of which is which is incorporated in its entirety herein by reference). For example, in some embodiments, a LSR construct and / or a CAR construct comprises an oligomerization element for the purpose of coordinating protein oligomerization and subsequent signal transduction. In some embodiments, a LSR construct and / or a CAR construct comprises a homodimerization, heterodimerization, trimerization, tetramerization, or oligomerization element, or combinations thereof, in order to achieve oligomerization. Exemplary oligomerization elements that may be used in accordance with certain embodiments of the present disclosure are described in, for example, Engel & Kammerer, Page 50 of 89 12923515v1Docket No.: 2017422-0007 2000, Thomas et al., 2013, and Mittl et al., 2000, the contents of which are hereby incorporated by reference in its entirety. In some embodiments, an oligomerization element is or comprises a short flexible peptide linker which joins the light and heavy chains of a scFV as described in, for example, Ahmad et al., 2012, the contents of which are hereby incorporated by reference in its entirety.

[0178] Those skilled in the art, reading the present disclosure will appreciate that activation of a LSR and CAR expressed in provided cells (e.g., immune cells, e.g., T cells) can be via the same mechanism or different mechanisms described herein. LSR-Expressing Cells

[0179] Among other things, the present disclosure provides preparations of cells engineered to express a LSR construct as described herein. Alternatively or additionally, the present disclosure provides preparations of cells engineered to express a LSR construct, and a CAR construct as described herein.

[0180] In some embodiments, provided cells (e.g., provided cell preparations) are immune cells. In some embodiments, provided cells (e.g., provided cell preparations) are T cells. In some embodiments, provided cells (e.g., provided cell preparations) are NK cells. In some embodiments, provided cells (e.g., provided cell preparations) are macrophages. In some embodiments, provided cells (e.g., provided cell preparations) are T cells that express a TCR. In some embodiments, such TCR is a natural TCR; in some embodiments, such TCR is an engineered TCR. In some embodiments, provided cells (e.g., provided cell preparations) are T cells that express a CAR. In some embodiments, provided cells (e.g., provided cell preparations) are T cells that express a CAR, wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, e.g., CD3Z or CD3C) activating element (e.g., zlessCAR). In some embodiments, provided cells (e.g., provided cell preparations) are T cells that express a CAR, wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, e.g., CD3 Z or CD3C) activating element, and a LSR. In some embodiments, provided T cells express a CAR targeting a tumor surface antigen, wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, e.g., CD3Z or CD3C) activating element, and a LSR targeting a lysis-associated antigen (e.g., an intracellular antigen). Page 51 of 89 12923515v1Docket No.: 2017422-0007

[0181] In some embodiments, provided cells (e.g., provided cell preparations) are T cells that comprise a combination of an LSR described herein and a CAR described herein. In some embodiments, said LSR and CAR may comprise the same transmembrane elements. In some embodiments, said LSR and CAR may comprise different transmembrane elements. In some embodiments, said LSR and CAR may comprise the same signaling elements. In some embodiments, said LSR and CAR may comprise different signaling elements.

[0182] In some embodiments, provided cells (e.g., provided cell preparations) are T cells that express an anti-mesothelin CAR, wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, e.g., CD3Z or CD3C) activating element, and a KRAS- targeting LSR.

[0183] In some embodiments, provided cells (e.g., provided cell preparations) are cells that have been cultured. In some embodiments, provided cells (e.g., provided cell preparations) are cells that have been isolated from a subject and cultured (e.g., expanded) ex vivo. In some such embodiments, provided cells are T cells, such as TIL cells.

[0184] In some embodiments, provided cells (e.g., provided cell preparations) include a plurality of different T cells (e.g., a plurality of T cells expressing different TCRs), each of which has been engineered to express the same LSR. Among other things, the present disclosure provides an insight that provided cell preparations will maintain their endogenous TCR heterogeneity.

[0185] In some embodiments, provided cells (e.g., a provided preparation of cells) is expanded from a sample obtained from a subject. In some embodiments, such sample is or comprises blood or other bodily fluid, or a tissue sample. In some embodiments, a sample is obtained from a site that is in the vicinity of a tumor. In some embodiments, such site comprises part or all of a tumor. In some embodiments, a sample is or comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, a sample is or comprises bone marrow. In some embodiments, a sample is or comprises a spleen, lymph node, or thymus sample. In some embodiments, a sample is or comprises tumor tissue (e.g., that may comprise TILs).

[0186] Those skilled in the art, reading the present disclosure, would be aware of appropriate source(s) of sample(s) for particular applications as described herein. Page 52 of 89 12923515v1Docket No.: 2017422-0007

[0187] In some embodiments, a population of cells (e.g., T cells) is obtained from a subject and engineered to express a LSR and a CAR, wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, e.g., CD3Z or CD3C) activating element. In some embodiments, a population of cells (e.g., T cells) is isolated from a patient to whom the engineered population of cells is to be administered.

[0188] In some embodiments, a population of T cells is obtained. In some embodiments, cells of an obtained population express T cell receptor(s). In some embodiments, cells of an obtained population of T cells are engineered to alter expression of their natural TCR (e.g., to replace it with an engineered TCR) and / or to express a CAR. In some embodiments, a population of T cells is engineered according to methods described by, for example, Ellis et al., 2021, the contents of which are hereby incorporated by reference in its entirety.

[0189] In some embodiments, a method for producing an engineered population of T cells is transduction with a viral vector or a polynucleotide construct comprising a coding sequence for a LSR and a CAR. Those skilled in the art will be familiar with various technologies for engineering cells (e.g., to express LSRs and / or engineered CARs and / or TCRs, etc.) in accordance with the present disclosure. For example, technologies such as electroporation, particle (e.g., gold particle, lipid nanoparticle, etc.) delivery, transfection (e.g., chemical transfection), vesicle (e.g., exosome or other lipid vesicle) delivery, viral delivery, etc. are available (see, for example, Alzubi et al., 2021, Paunovska et al., 2022). Additionally or alternatively, examples of methods for preparation of cells for engineering and methods for engineering cells (e.g., to express LSRs and / or engineered CARs and / or TCRs, etc.) in accordance with the present disclosure that are known in the art include, for example, those described in U.S. Pat. No.11,266,739 B2, the contents of which are hereby incorporated by reference in its entirety.

[0190] Alternatively or additionally, the present disclosure describes a method of generating a population of engineered cells (e.g., immune cells) comprising introducing an in vitro transcribed polynucleotide construct (e.g., RNA or synthetic RNA) into a cell, wherein the polynucleotide construct encodes a LSR construct as described herein (see, for example, U.S. Pat. No.11,453,719 B2, which is herein incorporated by reference in its entirety). Page 53 of 89 12923515v1Docket No.: 2017422-0007

[0191] Among other things, the present disclosure provides methods for preparing a population of cells engineered to express a LSR construct and a CAR construct, wherein the signaling element of the CAR does not comprise a CD3 zeta (CD3 Z or CD3C) activating element ,as described herein for administering to a subject. In some embodiments, an engineered population of T cells may be expanded and activated as described in U.S. Pat. No.11,084,880 B2, or references cited therein, each of which is herein incorporated by reference in its entirety. Expression of a LSR construct and / or a CAR construct in an engineered population of T cells may be evaluated by ways known in the art including but not limited to, Western Blot analysis using an antigen for a lysis-associated-antigen binding moiety, an antibody against a co-stimulation element(s), and any combination thereof, and analysis of in vitro expansion of engineered zlessCAR / LSR-expressing T cells by flow cytometry.

[0192] Among other things, the present disclosure describes assays for measuring activity of a population of T cells engineered to express a LSR construct and a CAR construct, wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, e.g., CD3Z or CD3C) activating element, as described herein, including but not limited to anti-cancer activity in in vitro and animal models. Applications / Uses Methods of treatment

[0193] Among other things, in some embodiments, technologies of the present disclosure are used to treat a disease, disorder, or condition. In some embodiments, such diseases may include tumors, cancers, or other proliferative diseases, and / or infectious agents or disease. In some embodiments, provided herein are methods treating a disease comprising administering a population of cells engineered to express a LSR construct as described herein that targets a lysis-associated antigen (e.g., an intracellular lysis-associated antigen) produced by diseased cell(s) in the subject and a CAR, wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, e.g., CD3Z or CD3C) activating element (e.g., zlessCAR).

[0194] In some embodiments, a method of treatment comprises administration of at least two successive doses of zlessCAR / LSR-expressing cells (e.g., cells engineered to express an Page 54 of 89 12923515v1Docket No.: 2017422-0007 LSR and a CAR, wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, e.g., CD3Z or CD3C) activating element, as described herein). In some embodiments, a second dose is administered if (and optionally only if) an intervening assessment has determined that the subject continues to suffer from the relevant disease, disorder or condition and / or that the disease, disorder or condition progressed. Exemplary dosing regimens that may be useful in accordance with certain embodiments of the present disclosure are described in, for example, U.S. Pat. No.11,266,739 B2, incorporated in its entirety herein by reference.

[0195] In some embodiments, a subject who receives therapy with a preparation of engineered cells (i.e., zlessCAR / LSR-expressing cells) as described herein is also receiving other therapy for the disease, disorder or condition. For example, where the disease disorder or condition is or comprises cancer, such subject may receive provided therapy in combination with, for example, one or more other immune-oncology therapies such as, for example other adaptive cell therapy, e.g., targeting one or more disease-associated antigens and / or checkpoint inhibitor therapy. Alternatively or additionally, such subject may be receiving or have received cancer therapy that is or comprises chemotherapy, surgery and / or radiation therapy. In some embodiments, said other therapy for the disease, disorder or condition is administered prior to a therapy with a preparation of engineered cells (i.e., zlessCAR / LSR-expressing cells) as described herein.

[0196] In some embodiments, a method of treating a disease, disorder, or condition comprises a priming event. In some embodiments, a priming event is other immune- oncology therapies such as, for example other adaptive cell therapy, chemotherapy, radiation therapy, and / or checkpoint inhibitor therapy. In some embodiments, a priming event is infusion of a lysis-associated antigen. Regulating administered cells

[0197] Among other things, in some embodiments, the present disclosure describes technologies for regulating zlessCAR / LSR-expressing cells (e.g., immunosuppressive agents, small molecule drugs, etc.) described herein, as described by, for example, Brandt et al.2020, Park et al., 2021, the contents of which is hereby incorporated by reference herein in its entirety. Page 55 of 89 12923515v1Docket No.: 2017422-0007

[0198] As described herein, cytokine release syndrome (CRS) is a well-known consequence of CAR-T therapy. It is an insight of the present disclosure that methods developed to regulate CAR T activity may be used to regulate activity of zlessCAR / LSR- expressing cells described herein. In some embodiments, activity of a zlessCAR / LSR- expressing cell (e.g., engineered T cell) is inhibited by administering immunosuppressive agents (e.g., corticosteroids).

[0199] As described herein, in some embodiments, a LSR construct may comprise a masking element, for example, that may be cleaved off or moved in a tumor microenvironment. Such a LSR construct comprises a masking element inhibiting activity of a lysis-associated-antigen binding moiety, with a linker sensitive to proteolytic cleavage. In such embodiments, activity of zlessCAR / LSR-expressing cells is regulated by tumor- associated proteases removing a masking peptide in the tumor microenvironment.

[0200] In some embodiments, a lysis-associated-antigen binding moiety is or comprises a conditional single chain variable fragment (scFv), wherein the scFv comprises a small molecule-based control element, so that LSR activity can be regulated by administration of a small molecule drug. In such embodiments, affinity of a lysis-associated-antigen binding moiety towards a target protein is reduced by activity of a small molecule drug.

[0201] In some embodiments, a LSR construct may comprise a virally derived NS3 protease and a degradation element such that the LSR is tagged for degradation. Moreover, activity of such a LSR is regulated by administration of a NS3 protease inhibitor.

[0202] As described herein, in some embodiments, a CAR construct may comprise a masking element, for example that may be cleaved off or moved in a tumor microenvironment. Such a CAR construct comprises a masking element inhibiting activity of an antigen binding moiety, with a linker sensitive to proteolytic cleavage. In such embodiments, activity of zlessCAR / LSR-expressing cells is regulated by tumor-associated proteases removing a masking peptide in the tumor microenvironment.

[0203] In some embodiments, an antigen binding moiety is or comprises a conditional single chain variable fragment (scFv), wherein the scFv comprises a small molecule-based control element, so that CAR activity can be regulated by administration of a small molecule Page 56 of 89 12923515v1Docket No.: 2017422-0007 drug. In such embodiments, affinity of a lysis-associated-antigen binding moiety towards a target protein is reduced by activity of a small molecule drug.

[0204] In some embodiments, a CAR construct may comprise a virally derived NS3 protease and a degradation element such that the CAR is tagged for degradation. Moreover, activity of such a CAR is regulated by administration of a NS3 protease inhibitor.

[0205] As described herein, zlessCAR / LSR-expressing cells may comprise an adapter CAR. Activity of such zlessCAR / LSR-expressing cells can be regulated via the use of adapter molecules. In some embodiments, timing of zlessCAR / LSR-expressing cell activity can be modulated via timing of adapter molecule administration. In some embodiments, simultaneous and sequential combinatorial targeting of different tumor antigens can be achieved via adaptor molecules and zlessCAR / LSR-expressing cells comprising an adapter CAR. EXEMPLIFICATION Example 1: Exemplary Lysis Sensor Receptor Constructs Targeting Lysis-Associated- Antigens

[0206] The present Example provides exemplary compositions, preparations, constructs, virions, preparation of virions, and host cells for gene therapy and related methods that target an intracellular product of diseased cells.

[0207] The present disclosure provides an insight that connecting T cell activation to presence of lysed / lysing cells could dramatically improve engineered T cell therapy (e.g., CAR-T therapy). Among other things, the present disclosure provides CAR constructs that respond specifically to cell lysis, described herein as a Lysis Sensor Receptor (LSR). As shown in FIG.1, a T cell (e.g., immune effector cell) comprising a LSR as described herein is activated by a lysis-associated-antigen(s) (e.g., an intracellular disease antigen, e.g., tumor antigen) released upon lysis of a target cell, resulting in T cell cytotoxic activity, which results in more T cell effector activation in a positive feedback loop. Exemplary LSR construct structures are depicted in FIG.2. In some embodiments, a LSR construct comprises an extracellular single chain variable fragment (scFv) of an antibody known to bind a relevant antigen of interest operably linked to a transmembrane element, and one or Page 57 of 89 12923515v1Docket No.: 2017422-0007 more T cell co-stimulation elements. In some embodiments, a LSR construct further comprises a CD3^ activation element.

[0208] Moreover, the present Example describes certain exemplary models for LSR activation by a lysis-associated-antigen in accordance with the present disclosure.

[0209] Among other things, the present disclosure describes that, in some embodiments, antigen binding leads to a direct conformational change in an intracellular domain of a LSR resulting in direct activation (as shown in FIG.3A). In some embodiments, antigen binding causes a change in interaction of a LSR with other endogenous proteins, resulting in signal activation (as shown in FIG.3B). In some embodiments, antigen binding causes dimerization or higher order oligomerization of a LSR through the binding of dimeric or oligomeric ligands (as shown in FIG.3C). In some embodiments, antigen binding causes dimerization through binding of multiple ligand molecules that form multimeric structures as a result of interactions with other macromolecules, such as nucleic acids, carbohydrates, lipids or proteins (as shown in FIG.3D). In some embodiments, an antigen brings LSRs that recognize different antigenic regions of a single protein or a protein complex together resulting in signal activation (as shown in FIG.3E). Example 2: Exemplary Lysis Sensor Receptor Constructs Targeting KRAS

[0210] The present Example describes certain exemplary Lysis Sensor Receptor (LSR) constructs in accordance with the present disclosure.

[0211] Anti-KRAS antibodies have been reported in certain cancer patients (see, for example, Collins & Pasca Di Magliano 2014, the contents of which is hereby incorporated by reference herein in its entirety). The present disclosure provides an insight that, as KRAS is predominantly an intracellular protein, the observation of anti-KRAS antibodies in cancer patients may indicate that sufficient KRAS can be released from lysed cells in such patients that their immune systems can mount some level of successful response. The present disclosure harnesses this insight by providing LSR constructs, and engineered T cell populations that contain or express LSR constructs, targeting KRAS.

[0212] For example, the present Example describes KRAS-targeting LSR construct(s) in which KRAS-biding-element(s) of a human anti-KRAS antibody (e.g., as may have arisen in Page 58 of 89 12923515v1Docket No.: 2017422-0007 a cancer patient) are linked to a transmembrane element and a T cell receptor co-stimulation element. The present Example specifically exemplifies LSR construct(s) in which KRAS- binding-element(s) of a human anti-KRAS antibody (e.g., as described in Kim et al., 2018, the contents of which is hereby incorporated by reference herein in its entirety) are included in a single-chain antigen-binding domain (scFv) that is linked to a transmembrane element and one or more co-stimulation elements, as shown in FIG.4. In some embodiments, a LSR construct described herein comprises a KRAS-binding-element linked to a transmembrane element and CD28, OX40, and CD3^ co-stimulation elements. Generation of LSR Constructs

[0213] The present Example describes generation of LSR constructs for use in accordance with the present disclosure. Constructs comprising a coding sequence encoding a LSR described herein are generated and tested in producer cells according to standard protocols. In some embodiments, one or more LSR backbone constructs are designed for cloning into a lentiviral vector. In some embodiments, a preparation of virions comprising a LSR construct are produced by transfecting 293-T cells with a plasmid comprising a LSR construct described herein and three helper plasmids comprising a gag-pol gene, a rev gene, and a VSV-g envelope gene. Methods for production of a lentiviral vector that may be utilized in accordance with some embodiments of the present disclosure are described in, for example, Merten et al., 2016.

[0214] In some embodiments, a LSR construct is cloned into an AAV vector. In some embodiments, 293 cells are transfected with a plasmid comprising a LSR construct, an AAV helper plasmid, and an adenoviral helper plasmid. Methods for production of an AAV vector in accordance with the present disclosure are described in, for example, Shin et al.2013.

[0215] Accordingly, in some embodiments, the present disclosure describes compositions, preparations, constructs, virions, population of virions, and host cells comprising a variant protoparvovirus VP1 capsid polypeptide can exhibit increased VP1 initiation relative to a reference VP1 capsid polypeptide. Page 59 of 89 12923515v1Docket No.: 2017422-0007 Expression of LSR Constructs on T Cells

[0216] In some embodiments, a population of T cells is obtained from healthy human donors and transduced with a preparation of virions comprising a KRAS-targeting LSR described herein. Expression of a KRAS-targeting LSR may be determined by flow cytometry. A population of T cells is cultured in RPMI 1640 medium with 10% FBS and stimulated with anti-CD3 / anti-CD28 Dynabeads (Invitrogen). T cells are transduced with a preparation of virions 24 hours after stimulation. Mock transduced cells are used as a negative control and T cells transduced with a CAR construct known in the art is used as a positive control. 4-6 days after transduction, expression of a LSR on the surface of an engineered T cell is evaluated by flow cytometry.

[0217] The activation of a LSR, such as through KRAS binding, requires the ability of KRAS to oligomerize an LSR. KRAS is post-translationally modified by myristoylation, the covalent addition of a 14-carbon unsaturated fatty acid. Myristoylation leads to the attachment of KRAS to the inner cell membrane where it is required to be localized for activity. Because multiple KRAS molecules are found localized to the inner cell membrane, binding to LSRs will lead to oligomerization of LSRs, as shown in FIG.5, resulting in activation of LSR signaling and stimulation of T cells.

[0218] It is an insight of the present disclosure that altering the composition of the intracellular domains of LSRs can modulate their ability to stimulate T cell activity. In some embodiments, an exemplary LSR construct comprises a co-stimulation element comprising a CD28 and an OX40 co-stimulation element as described herein. Among other things, the present example recognizes that lack of a CD3 element in a LSR construct can reduce stimulatory activity, resulting in reduction of systemic toxicity. In some embodiments, a co- stimulation element comprising a CD3 element can be used in combination with a LSR that is highly specific to mutant forms of an intracellular product of diseased cells (e.g. KRAS) as systemic activation through binding of a LSR is expected to be low.

[0219] The present Example specifically describes LSR constructs that target KRAS. Those skilled in the art, reading this Example and particularly in the context of the present disclosure will appreciate that analogous strategies can be implemented to provide LSR constructs that target alternative markers (e.g., alternative intracellular markers). Page 60 of 89 12923515v1Docket No.: 2017422-0007

[0220] For example, as shown in FIG.6, a number of oncogenes are mutated in a high percentage of tumors; several of the proteins encoded by these oncogenes are intracellular proteins. In some embodiments, the present disclosure provides LSR constructs (and associated technologies, as described herein) that target other intracellular oncogene- encoded proteins. To give but one example, a LSR comprising a lysis-antigen-binding moiety for one or more P53 epitopes is within the scope of this disclosure exemplified in FIG.7.

[0221] Furthermore, one feature of the present disclosure is that certain embodiments do not require that the target of an LSR be a disease-specific (or disease-associated) target. That is, because the event of cell lysis may be disease-associated (or disease-specific), in some embodiments, LSR constructs may be directed at intracellular target(s) that are present in non-diseased cells (e.g., at a level or frequency comparable to or even higher than that at which they are present in diseased cells). Thus, the present disclosure provides uniquely flexible targeting technologies. Example 3: T cells engineered to express a LSR can be activated by exogenous mutant KRAS

[0222] The present Example teaches that T cells engineered to express a LSR can be activated by exogenous mutant KRAS.

[0223] Without wishing to be bound to any theory, the present disclosure notes that chimeric antigen receptor (CAR) activation is dependent on antigen density, CAR density, and co-stimulation elements (see, for example, Majzner et al., 2020). The present Example demonstrates that engineered T cells expressing a KRAS-targeting LSR construct(s) show activation by exogenous mutant KRAS when stimulated by incubation with cell lysates from KRAS-mutant cell lines. In some embodiments a KRAS-mutant cell line corresponds to a G12D KRAS mutant (e.g., AsPC-1, HPAF-II (pancreatic), GP2d, LS180 (colon), T3M-10 (lung). In some embodiments, a KRAS-mutant cell line corresponds to a G12V KRAS mutant (e.g., Capan-1, KP-3 (pancreatic), SW480 (colon), COLO 668 (lung). In some embodiments a KRAS-mutant cell line corresponds to a G12C KRAS mutant (e.g., MIA PaCa-2 (pancreas), SW837 (colon), Calu-1, LU65 (lung). In some embodiments, a KRAS- Page 61 of 89 12923515v1Docket No.: 2017422-0007 mutant cell line corresponds to a KRAS variant with a mutation at a position other than amino acid 12.

[0224] KRAS-targeting LSR-expressing T cells or control T cells are expanded until the end of log-phase growth, they are artificially stimulated overnight and subsequently co- cultured for 16 hours with either lysed AsPC-1-KRAS specific target cells, lysed non-KRAS target cells as positive control or no target cells as negative control at a 3 to 1 ratio of effector cells to target cells. Culture supernatants are to be harvested to measure T cell proliferation in response to co-culture conditions and cytokine production. IFN-gamma and IL-2 concentration is measured by specific ELISA following manufacturer instructions (R&D). T cells engineered to express a KRAS-targeting LSR will show increased cytokine production after co-culturing with KRAS mutant cells lines when compared to engineered T cells co-cultured with non-KRAS-mutant cells lines. Cytotoxic activity of KRAS-targeting LSR-expressing-T cells is to be evaluated using a Cr release-assay. Alternatively or additionally, the present disclosure recognizes that methods of evaluating cytotoxic activity that may be used in accordance with the present disclosure are known in the art (e.g., flow cytometry, cell viability assays). KRAS-targeting LSR-expressing-T cells are able to induce lysis of KRAS-expressing cell lines with little activity towards non-KRAS-mutant cell lines demonstrating that KRAS LSR T cells are specifically activated by lysed tumor cells only when these cells have KRAS mutations. Example 4: T Cells Expressing a LSR Exert Antibody Dependent Cancer Cell Killing

[0225] The present Example provides exemplary constructs, preparations, population of engineered T cells, and producer cells for CAR-T therapy and related methods that demonstrate enhanced killing of a KRAS mutation containing cell line as described herein.

[0226] In one exemplary embodiment, anti-cancer activity of a KRAS-targeting LSR is evaluated in a preclinical animal model of pancreatic cancer using the cell line AsPC-1. AsPC-1 cells are engineered to express Click-Beetle Green Luciferase (CB-G Luc+) to track tumor progression by bioluminescent in vivo imaging (IVIS) and Living Image software (Perkin AElmer). CB-G Luc+AsPC-1 cells are cultured for four weeks and later IV injected into NSG recipients. Engineered T cells expressing the KRAS-targeting LSR are IV injected and tumor burden is evaluated by in vivo imaging. Page 62 of 89 12923515v1Docket No.: 2017422-0007

[0227] In the present Example, a KRAS-mutant cell line (e.g, AsPC-1) is grown and expanded in vitro according to standard protocols and IV injected in NGS mice. Mice are to be administered 5xl06T cells 7-8 days after tumor implantation. Cells are partially thawed in a 37 degree Celsius water bath and then completely thawed by addition of 1 ml of cold sterile PBS to the tube containing the cells. A population of thawed cells is transferred to a 15 ml falcon tube and adjusted to a final volume of 10 mLs with PBS. KRAS-targeting LSR T cells are washed twice at 1000 rpm for 10 minutes each time and then counted on a hemocytometer. KRAS LSR T cells are normalized for transduction so that all mice are treated with the same percentage of cells, and resuspended at a concentration of 50x106 cells per mL of cold PBS and kept on ice until mice are dosed.

[0228] Five to seven mice per group are to be treated with either 100uL of KRAS- targeting LSR T cells, untransduced T cells (mock), or PBS alone. Mice are monitored according to standard protocols for health status, including body weight measurements, and tumor burden via in vivo imaging. Numbers of KRAS LSR T cells are also evaluated via peripheral blood FACS analysis. Mice treated with KRAS LSR T cells demonstrate expansion of KRAS LSR T cells compared to mock treated controls. In addition, KRAS LSR T cell treated mice show significant reduction in tumor growth compared to controls. Lack of efficacy seen with untransduced T cells (non-lysis-associated-antigen targeting T cells) correlates with a reduced change in tumor growth compared to KRAS LSR T cell treated mice. Example 5: Exemplary Dosing Regimen

[0229] The present Example provides exemplary constructs, preparations, population of engineered T cells, and producer cells for LSR T therapy as described herein. Additionally, the present Example provides exemplary dosing regimens for LSR T therapy as described herein.

[0230] Among other things, the present disclosure describes steps of a method of administering a LSR T cell therapy. Methods for administering CAR-T cell therapy known in the art may be used in accordance with the present disclosure. As shown in FIG.8, exemplary steps of administering a CAR therapy (e.g., a LSR therapy as described herein) include, (1) collecting a patient’s peripheral blood, (1) isolating a population of T cells from Page 63 of 89 12923515v1Docket No.: 2017422-0007 a blood sample, (3) activation and amplification of such a population of T cells, (4) engineering the obtained population of T cells to express a designed CAR, (5) amplification and quality control measures of an engineered population of CAR-expressing T cells, and (6) transfusion of engineered CAR-T cells in the patient from whom the population of T cells was obtained.

[0231] As of 2017, six CAR-T therapies have been approved: Abecma (idecabtagene vicleucel), Breyanzi (lisocabtagene maraleucel), Kymriah (tisagenlecleucel), Tecartus (brexucabtagene autoleucel), Yescarta (axicabtagene ciloleucel), Carvykti (ciltacabtagene autoleucel). The dosing regimens for these approved therapies are attached as figures herein.

[0232] As can be seen in FIGs.9-14, all of these dosing regimens involve a step of administering a lymphodepleting regimen before infusion of a population of engineered T cells. In some embodiments, a lymphodepleting regimen is cyclophosphamide and fludarabine. All of these dosing regimens involve not using a leukodepleting filter, verifying a subject’s identity prior to infusion, premedication with acetaminophen and an H1 antihistamine, confirming availability of tocilizum prior to infusion, and dosing of a CAR-T therapy based on the number of CAR-positive viable T cells. All of these dosing regimens are for autologous use only. All of these dosing regimens are for intravenous use only. Example 6: Production of Exemplary Lysis Sensor Receptor Constructs Targeting KRAS

[0233] The present Example documents expression of KRAS-targeting LSRs as described herein in T cells.

[0234] Two exemplary LSR constructs were designed and produced using a KRAS scFv (specifically, an scFv targeting mutant KRAS; the amino acid sequence of this scFv is provided as SEQ ID NO: 1, and is based on human KRAS antibody 1F4_4F10, as described in U.S. Patent No.11,174,314, which is herein incorporated by reference in its entirety), as depicted in FIG.15. Specifically, as indicated in FIG.15, LSR Construct 1 includes CD28 and 4-1BB co-stimulation elements and a CD3^ activating element; LSR Construct 2 includes only the CD28 and 4-1BB co-stimulation elements. Each of these constructs further includes a Flag polypeptide tag used to assess expression in T cells. Those skilled in Page 64 of 89 12923515v1Docket No.: 2017422-0007 the art will be familiar with such tags; the particular Flag tag (aka FLAG octapeptide; FLAG epitope) used in exemplified LSR Constructs 1 and 2 is set forth in SEQ ID NO: 6.

[0235] LSR Constructs 1 and 2 were synthesized; their identities were confirmed by sequencing in both directions. They were each cloned into a pCD510 lentiviral vector (Systems Bioscience, Palo Alto, CA, USA). Lentivirus was generated using 293 FT cells, Lentivirus Packaging Mix and transfection agent (Alstem, Richmond, CA, USA) (e.g., as described in Berahovich et al., 2017, which is herein incorporated by reference in its entirety). Virus titers were determined by quantitative RT-PCR using a Lenti-X qRT-PCR kit (Takara Bio, Mountain View, CA, USA) according to manufacturer’s protocol and a 7900HT thermal cycler (Thermo Fisher Scientific, South San Francisco, CA, USA). Lentiviral titers were expressed in pfu / mL and ranged 1–10 × 108pfu / mL.

[0236] Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque solution (GE Healthcare, Chicago, IL). PBMCs were suspended at 1 × 106cells / mL in AIM V-AlbuMAX medium (Thermo Fisher) containing 10% FBS with 300 U / mL IL-2 (Thermo Fisher). PBMC were activated with an equal number of CD3 / CD28 Dynabeads (Thermo Fisher), and cultured in non-treated 24-well plates. At 24 and 48 h, lentivirus was added to cultures at a multiplicity of infection (MOI) of 5 with 1 μL of TransPlus transduction enhancer (AlStem). As a control for transduction efficiency, the vector encoding the KRAS LSR also contained the polynucleotide encoding green fluorescent protein (GFP). KRAS-targeting LSR-expressing T cells were counted every two to three days and fresh medium with 300 U / mL IL-2 was added to cultures to maintain a cell density at 1 × 106cells / mL.

[0237] To measure KRAS-LSR expression, 5 × 105cells were suspended in 100 μL of buffer (1× PBS with 0.5% BSA+ 0.5 mM EDTA + 0.1% NaN3) and incubated on ice with 1 μL of human serum (Jackson Immunoresearch, West Grove, PA, USA) for 10 min. Anti- FLAG APC (Biolegend; 637308) was used to assess expression of FLAG-Tagged KRAS LSR. Cells were rinsed with buffer and acquired on an Agilent NovoCyte 300 Flow cytometer. Cells were analyzed first for light scatter versus GFP staining, then GFP−live gated cells were plotted for APC staining. FIG.16 depicts flow cytometry data indicating detection of KRAS-LSR-T cells expressing GFP and LSR Construct 1. Page 65 of 89 12923515v1Docket No.: 2017422-0007

[0238] Accordingly, the present Example documents expression of KRAS-LSRs as described herein in T cells. Example 7: Expression of KRAS-LSR Increases T-Cell Killing

[0239] The present Example documents successful production of T cells expressing both a LSR and CAR in accordance with the present disclosure. Moreover, the present Example confirms that a KRAS-targeting LSR (e.g., KRAS-LSR) enhances cell killing activity of CAR-T cells. In fact, the present Example confirms that a KRAS-LSR increased the rate and extent of cell killing by two different types of CAR-T cells: Meso-CAR-T cells and EpCAM-CAR-T cells.

[0240] T cells expressing a KRAS-targeting LSR (i.e., KRAS-LSR-T cells) were created as described in Example 6, expressing LSR Construct 1. Mesothelin-specific CAR-T cells (e.g., Meso-CAR-T cells) were created using a similar method by transducing T cells with lentivirus comprising a polynucleotide construct encoding a mesothelin-specific CAR (PMC958 ProMab Biotechnologies). T cells expressing both the KRAS-LSR and the Meso- CAR were created by simultaneously transfecting T cells with lentivirus comprising a polynucleotide encoding the KRAS-LSR construct and lentivirus comprising a polynucleotide encoding the Meso-CAR at a 4:1 ratio.

[0241] KRAS-LSR expression was measured using the FLAG-tag as described in Example 6. To measure Meso-CAR expression, cells were suspended in 100 µL of buffer 1× PBS with 0.5% BSA+ 0.5 mM EDTA + 0.1% NaN3 and incubated on ice with 1 µL of human serum for 10 min. Biotin-human Mesothelin protein (Acro Biosystems; MSN- H82E9) was added to the cells and incubated for 30 min at 4 ◦C, and after washing, PE- conjugated streptavidin (Biolegend; 405204) was added at 1:100 dilution for 30 min incubation at 4 ◦C. Cells were rinsed with 3 mL of washing buffer, then stained for 10 min with 7-AAD (Biolegend;420404), suspended in buffer, and acquired on an Agilent NovoCyte 300 Flow cytometer. As depicted in FIG.17, 7% of transduced T cells showed expression of both a KRAS-LSR and a Meso-CAR.

[0242] Those skilled in the art will appreciate that mesothelin expression has been described as associated with a variety of different solid tumors, including, for example, mesotheliomas, epithelial ovarian cancers, and pancreatic adenocarcinomas, and also in lung Page 66 of 89 12923515v1Docket No.: 2017422-0007 and uterine malignancies as well as cholangiocarcinoma. See, for example, Pende, Cancers 14:1550, 2022, and references cited therein. The present Example documents effects of provided KRAS-targeting LSR with a Mesothelin-targeting CAR-T in human ovarian cancer cells.

[0243] Specifically, adherent A1847 target cells (e.g., human ovarian cancer cells) were seeded into 96-well E-plates (Acea Biosciences, San Diego, CA, USA) at 1 × 104 cells per well and monitored in culture overnight with impedance-based real-time cell analysis (RTCA) iCELLigence system (Acea Biosciences). After 24 hours, medium was removed and replaced with AIM V-AlbuMAX medium containing 10% FBS ± 1 × 105 effector cells: KRAS-targeting LSR-expressing T cells, Meso-CAR-T cells, or T cells that were transduced with both an LSR and CAR construct. Cells were monitored with a RTCA system, and impedance was plotted over time. CAR-T cell killing was calculated as (impedance of target cells with mock-transfected T cells—impedance of target cells with effector cells) × 100 / impedance of target cells with mock transfected T cells.

[0244] T cells expressing both a KRAS-LSR and Meso-CAR showed higher percent cell killing of A1847 cells (e.g., human ovarian cancer cells) relative to Meso-CAR-T cells. FIG.18 depicts that KRAS-LSR-T cells had no detected cell killing activity on their own, whereas Meso-CAR showed about 40% cell killing at 20 hours. KRAS-LSR / Meso-CAR-T cells showed about 65% cell killing at 20 hours. FIG.18 documents that co-expression of a LSR and CAR led to increased cell killing relative to Meso-CAR alone, thus documenting a synergistic effect of such co-expression. Moreover, as also shown in FIG.18, LSR-boosted cell killing activity was delayed by about 5 hours, consistent with LSR activation following initial cell killing and release of intracellular KRAS.

[0245] Without wishing to be bound to any theory, FIG.19 shows a schematic depicting a proposed mechanism of action for increased T cell activation and increased cell killing by an exemplary KRAS LSR.

[0246] Dual CAR-T cells expressing a KRAS-LSR and an epithelial cell adhesion molecule-specific CAR (e.g., EpCAM-CAR) (ProMab Biotechnologies, PMC1057) were also produced as described herein. EpCAM-CAR expression was assessed using an anti- Page 67 of 89 12923515v1Docket No.: 2017422-0007 mouse FAB PE-conjugated antibody. As shown in FIG.20, 4% of transduced T cells showed expression of both a KRAS-LSR and an EpCAM-CAR.

[0247] Those skilled in the art will appreciate that EpCAM expression has been described as a biomarker of cancer stem cells (CSCs) or circulating tumor cells (CTCs) and as associated with ovarian cancer, pancreatic cancer, and adenocarcinomas of various primary sites. See, for example, Spizzo, Journal of Clinical Pathology.64(5): 415–420, 2011, and Liu, Exp Hematol Onco.l 11, 97, 2022, and references cited therein, each of which is herein incorporated by reference in their entirety.

[0248] The present Example documents effects of provided KRAS-targeting LSR with a EpCAM-targeting CAR-T in human pancreatic cancer cells. Specifically, T cells expressing both a KRAS-LSR and an EpCAM-CAR showed higher percent cell killing of SW-1990 cells (e.g., pancreatic cancer cells, e.g., pancreatic adenocarcinoma cells) relative to EpCAM-CAR-T cells alone, as depicted in FIG.21.

[0249] Accordingly, the present Example confirms increased killing due to LSR construct expression in CAR-T targeting two different tumor antigens; these tumor antigens are associated with different types of tumors. Those skilled in the art will be aware of a variety of tumor antigens against which CAR-T have been or can be developed, and will appreciate the usefulness of provided technologies in the context of such CAR-T. For example, several CD19-specific CAR-T and BCMA-specific CAR-T cell therapies have been FDA approved for treatment of Acute Lymphoblastic Leukemia, multiple myeloma, and different B cell malignancies (Mitra, Front Immunol.14:1188049, 2023, which is herein incorporated by reference in its entirety). Example 8: Exemplary KRAS-LSR-T cells Showed Increased T Cell Activation

[0250] The present Example documents that co-expression of an exemplary LSR and a CAR in T cells, as described herein, results in a higher degree of T cell activation relative to expression of just the CAR in T cells.

[0251] A1847 cells (e.g., human ovarian cancer cells) were cultured with effector cells, Meso-CAR-T cells, Meso-CAR / KRAS-LSR-T cells (i.e., T cells expressing both a KRAS- targeting LSR and a Meso-CAR, or non-transduced T cells, at a effector to target ratio or Page 68 of 89 12923515v1Docket No.: 2017422-0007 E:T ratio of 5:1 or 10:1 in U-bottom 96-well plates with 200 μL of AIM V-AlbuMAX medium containing 10% FBS, in triplicate. After 24 hours a top 150 μL of medium was transferred to V-bottom 96-well plates and centrifuged at 300 g for 5 min to pellet any residual cells. Supernatant was transferred to a new 96-well plate and analyzed by ELISA for IFN-gamma levels using a kit from Thermo Fisher (South San Francisco, CA, USA) according to manufacturer’s protocol. As depicted in FIG.22, Meso-CAR / KRAS-LSR-T cells have a higher degree of T cell activation, as measured by IFN-gamma expression, relative to Meso-CAR-T cells alone, and relative to a mixture of Meso-CAR-T cells and KRAS-LSR-T cells.

[0252] Accordingly, the present Example documents that T cells expressing both a LSR and a CAR have a higher degree of T cell activation relative to T cells expressing just the CAR (i.e., CAR-T cells). Specifically, the present Example documents that Meso- CAR / KRAS-LSR-T cells have a higher degree of T cell activation relative to Meso-CAR-T cells alone, and relative to a mixture of Meso-CAR-T cells and KRAS-LSR-T cells.

[0253] Those skilled in the art, reading the present Example, will appreciate that the particular LSR and CAR constructs utilized (i.e., KRAS-targeting LSRs, Meso-CARs, and EpCAM-CARs) are exemplary and not limiting of the present disclosure; its findings can be expected to be applicable to LSRs targeting other antigens and / or to different CARs. Example 9: Exemplary LSR Show Increased Tumor Specificity and Without Increased Non-specific Activity

[0254] The present Example documents that T cells (e.g., CAR-T cells) expressing a KRAS-targeting LSR as exemplified herein (referred to in the present Example as “KRAS- LSR-T cells”) show tumor-specific efficacy (e.g., increased tumor-specificity relative to comparable CAR-T cells not expressing the KRAS-LSR-T cells) without increasing non- specific activity.

[0255] KRAS-LSR-T cells, Meso-CAR-T cells, and dual KRAS-LSR / Meso-CAR-T cells were prepared as described herein. Killing of A1847 cells was followed by RTCA. E:T ratio for all conditions was maintained at 10:1. For a mixed LSR + CAR sample, both the KRAS LSR and the meso CAR were added at a 5:1 E:T ratio. For all other samples the CAR Page 69 of 89 12923515v1Docket No.: 2017422-0007 or LSR cells were added at a 5:1 E:T ratio with an equivalent amount of mock transduced T cells used to bring an overall E:T ratio to 10:1.

[0256] As indicated in FIG.22, dual KRAS-LSR / Meso-CAR-T cells increased activation as measured by IFN-gamma secretion relative to Meso-CAR-T cells. Mixing the two populations of T cells, those with the LSR alone and those with the Meso-CAR alone, does not lead to increased T cell activation. Without wishing to be bound to any theory, the increase in cell activation in the dual KRAS-LSR / Meso-CAR-T cells is believed to be due to the close proximity of the KRAS LSR to the killed A1847 cells, whereas the lack of increased activation from mixing of the two populations of T cells is believed to be due to the lower concentration of KRAS in the overall supernatant.

[0257] Activation of KRAS-LSR-T cells by KRAS was further tested by addition of purified KRAS protein and A1847 cell lysates.1 ug / ml purified G12D KRAS (Acro Biosystems KRS-H51H4) was added to KRAS-LSR-T cells and cell killing of A1847 cells was followed using RTCA. Results were normalized to KRAS-LSR-T cells alone. Addition of purified KRAS did not increase cell killing in other controls (Data not shown). Supernatant from A1847 cells previously killed by Meso-CAR-T cells was collected and frozen. After thawing, supernatant was added to KRAS LSR-T cells at a 1:5 ratio and cell killing of A1847 cells was followed by RTCA. No cell killing was observed by addition of supernatant alone in other controls (Data not shown). Addition of supernatant from A1847 cells previously killed by Meso-CAR-T cells resulted in moderate cell killing of A1847 cells by KRAS-LSR-T cells, as depicted in FIG.23. Addition of purified G12D KRAS resulted in increased killing of A1847 cells by KRAS-LSR-T cells relative to addition of supernatant from A1847 cells previously killed by Meso-CAR-T cells, as depicted in FIG.23.

[0258] A KRAS antibody raised against mutant KRAS was used to test specificity of KRAS-LSR-T cell activation. Anti-KRAS mutant monoclonal antibody was used at a 1:1000 dilution (Thermofisher, cat#: MA5-36256). A1847 cell killing was followed by RTCA. As shown in FIG.24, anti-KRAS mutant antibody reduced rate of cell killing by dual KRAS LSR / Meso-CAR-T cells. Increased cell killing that results from dual expression of the KRAS-LSR and the Meso-CAR in the same T cells is blocked by a monoclonal antibody to mutant KRAS. Without wishing to be bound to any theory, the present disclosure describes that LSR activation occurs through binding of KRAS released by cell lysis. Page 70 of 89 12923515v1Docket No.: 2017422-0007

[0259] Accordingly, the present Example confirms that KRAS-LSR-T cells described herein show tumor-specific efficacy without an increase in non-specific activity. In particular, the present Example confirms that purified KRAS protein increases activity of a KRAS-LSR-T cell. The present Example also confirms that an anti-KRAS antibody inhibits activity of a KRAS-LSR-T cell. Example 10: Production of zlessCAR / LSR-Expressing Cells

[0260] The present Example describes production of immune cells that express a CAR, wherein the signaling element of the CAR does not comprise a CD3^ (CD3 zeta, CD3Z or CD3C) activating element, and a LSR as described herein. In particular, the present example describes T cells that express an anti-mesothelin zlessCAR (e.g., zlessMeso-CAR) and a KRAS-targeting LSR (e.g., KRAS-LSR).

[0261] As described herein, the present disclosure identifies challenges with existing CAR-T cell therapies in that most tumor cell surface antigens that have been identified or utilized as targets of such CAR-T cell therapies are also expressed on healthy cells, reducing tolerance to CAR-T therapy and limiting effectiveness. It is an insight of the present disclosure that pairing activation of a T cell on cell lysis, targeting a tumor surface antigen with a zlessCAR, wherein activity of the CAR is attenuated via lack of a CD3^ activation element, results in specific targeting of solid tumor cells.

[0262] T cells expressing both a KRAS-LSR and a zlessMeso-CAR (i.e., zlessCAR / KRAS-LSR-T cells) are created by simultaneously transfecting T cells with lentivirus comprising a polynucleotide encoding a KRAS-LSR construct (LSR Construct 1) and lentivirus comprising a polynucleotide encoding a zlessMeso-CAR (CAR Construct 1) at a 4:1 ratio.

[0263] KRAS-LSR expression is measured using the FLAG-tag as described in Example 6. To measure Meso-CAR expression, cells are suspended in 100 µL of buffer 1× PBS with 0.5% BSA+ 0.5 mM EDTA + 0.1% NaN3 and incubated on ice with 1 µL of human serum for 10 min. Biotin-human Mesothelin protein (Acro Biosystems; MSN-H82E9) is added to the cells and incubated for 30 min at 4 ◦C, and after washing, PE-conjugated streptavidin (Biolegend; 405204) is added at 1:100 dilution for 30 min incubation at 4 ◦C. Cells are rinsed with 3 mL of washing buffer, then stained for 10 min with 7-AAD Page 71 of 89 12923515v1Docket No.: 2017422-0007 (Biolegend;420404), suspended in buffer, and acquired on an Agilent NovoCyte 300 Flow cytometer.

[0264] As described herein, those skilled in the art will appreciate that mesothelin expression has been described as associated with a variety of different solid tumors, including, for example, mesotheliomas, epithelial ovarian cancers, and pancreatic adenocarcinomas, and also in lung and uterine malignancies as well as cholangiocarcinoma. See, for example, Pende, Cancers 14:1550, 2022, and references cited therein.

[0265] The present Example documents effects of provided KRAS-targeting LSR with a zlessMesothelin-targeting CAR-T in human ovarian cancer cells.

[0266] Specifically, adherent A1847 target cells (e.g., human ovarian cancer cells) were seeded into 96-well E-plates (Acea Biosciences, San Diego, CA, USA) at 1 × 104 cells per well and monitored in culture overnight with impedance-based real-time cell analysis (RTCA) iCELLigence system (Acea Biosciences). After 24 hours, medium is removed and replaced with AIM V-AlbuMAX medium containing 10% FBS ± 1 × 105 effector cells: KRAS-targeting LSR-expressing T cells, zlessMeso-CAR-T cells, or T cells that were transduced with both a KRAS-targeting LSR and zlessMeso-CAR construct. Cells are monitored with a RTCA system, and impedance is plotted over time. CAR-T cell killing is calculated as (impedance of target cells with mock-transfected T cells—impedance of target cells with effector cells) × 100 / impedance of target cells with mock transfected T cells.

[0267] Among other things, it is an insight of the present disclosure that T cells expressing both a KRAS-LSR and a zlessMeso-CAR will show similar level of cell killing of A1847 cells (e.g., human ovarian cancer cells) relative to Meso-CAR-T cells which comprise a CD3^ activation element. Moreover, it is an insight of the present disclosure that T cells expressing both a KRAS-LSR and a zlessMeso-CAR will show no or reduced cell killing in the absence of lysed tumor cells. It is an insight of the present disclosure that T cells expressing both a KRAS-LSR and a zlessMeso-CAR will show no or reduced cell killing of healthy cells expressing a tumor antigen (e.g., mesothelin).

[0268] Without wishing to be bound to any theory, FIG.26 shows a schematic depicting a proposed mechanism of action for T cell activation by a tumor antigen and priming by a Page 72 of 89 12923515v1Docket No.: 2017422-0007 lysis-associated antigen and specific cell killing of tumor cells by an exemplary zlessCAR / LSR described herein.

[0269] Accordingly, the present Example describes increased solid tumor cell killing due to LSR construct expression in CAR-T targeting a tumor antigen. Those skilled in the art will be aware of a variety of tumor antigens against which CAR-T have been or can be developed, and will appreciate the usefulness of provided technologies in the context of such CAR-T. For example, several CD19-specific CAR-T and BCMA-specific CAR-T cell therapies have been FDA approved for treatment of Acute Lymphoblastic Leukemia, multiple myeloma, and different B cell malignancies (Mitra, Front Immunol.14:1188049, 2023, which is herein incorporated by reference in its entirety). Example 11: Production of Dual Expression zlessCAR / LSR Constructs

[0270] The present Example describes production of immune cells that express a CAR, wherein the signaling element of the CAR does not comprise a CD3^ (CD3 zeta, CD3Z or CD3C) activating element, and a LSR as described herein.

[0271] As described in Example 10, T cells expressing both a KRAS-LSR and a zlessMeso-CAR (i.e., zlessMeso-CAR / KRAS-LSR-T cells) are created by simultaneously transfecting T cells with lentivirus comprising a polynucleotide encoding a KRAS-LSR construct and lentivirus comprising a polynucleotide encoding a zlessMeso-CAR. Alternatively or additionally, T cells expressing both a KRAS-LSR and a Meso-CAR are created by transfecting cells with lentivirus comprising a polynucleotide encoding both a CAR and LSR on a single construct separated by a viral peptide sequence known as P2A (see, for example, He et al., Blood vol.135,10 (2020): 713-723.; Schneider et al., Science translational medicine vol.13,586 (2021): eabc6401.; US20240082302A1, the contents of each of which is hereby incorporated by reference herein in their entirety). FIG.27 shows a dual expression construct design of a zlessMeso-CAR / KRAS LSR dual expression construct and a Meso-CAR / KRAS LSR dual expression construct used in the present Example.

[0272] Constructs were synthesized as described in Example 6. KRAS-LSR expression is measured using the FLAG-tag as described in Example 6. Meso-CAR expression is measured as described in Example 10. Page 73 of 89 12923515v1Docket No.: 2017422-0007

[0273] Sequences of dual expression CAR / LSR constructs used in the present Example are shown below. P2 protease cleavage site is shown in bold.

[0274] Exemplary zlessMeso-CAR / KRAS-LSR dual expression polypeptide sequence (SEQ ID NO: 11) MALPVTALLLPLALLLHAARPASQVQLVQSGAEVEKPGASVKVSCKASGYTFTDYY MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRS DDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSLSA SVGDRVTITCRASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCLQTYTTPDFGPGTKVEIKLEKPTTTPAPRPPTPAPTIASQPLSL RPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRAKRSGSGATNFSLLKQA GDVEENPGPMLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGVVQPGRSLRLSCAASG FTSRHPGMHWVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLFVQ LSSLRPEDTAVYYCATSLYSSMDLWGQGTTVTVSSGSTSGSGKPGSGEGSTKGQSVV TQPPSVSAAPGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIPDR FSASKSGTSASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTVLDYKDDD DKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSL LVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0275] Exemplary Meso-CAR / KRAS LSR dual expression polypeptide sequence (SEQ ID NO: 12) MALPVTALLLPLALLLHAARPASQVQLVQSGAEVEKPGASVKVSCKASGYTFTDYY MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRS DDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSLSA SVGDRVTITCRASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCLQTYTTPDFGPGTKVEIKLEKPTTTPAPRPPTPAPTIASQPLSL RPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRSGSGATNFSLL KQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGVVQPGRSLRLSCA ASGFTSRHPGMHWVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTL FVQLSSLRPEDTAVYYCATSLYSSMDLWGQGTTVTVSSGSTSGSGKPGSGEGSTKGQ SVVTQPPSVSAAPGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGI PDRFSASKSGTSASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTVLDYK DDDDKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Page 74 of 89 12923515v1Docket No.: 2017422-0007

[0276] FIG.28 shows T cells that co-express a zlessMeso-CAR and a KRAS LSR (i.e., zlessMeso-CAR / KRAS-LSR-T cells), and a Meso-CAR and a KRAS LSR (i.e., Meso- CAR / KRAS-LSR-T cells) (KRAS-LSR: anti-Flag-APC; Meso-CAR: Mesothelin-FITC).

[0277] Accordingly, the present Example documents production of dual T cells expressing both a Meso-CAR and a KRAS-LSR. In particular, the present Example documents production of dual T cells expressing both a Meso-CAR and a KRAS-LSR via transduction with a lentivirus comprising a dual expression construct in which the CAR and LSR were separated by a P2 protease cleavage site. Example 12: Exemplary KRAS-LSR-T cells Showed Specific Activation by KRAS Mutant Protein Target

[0278] The present Example describes target specificity of LSR-expressing T cells described herein. In particular, the present Example demonstrates specific activation of zlessMeso-CAR / KRAS-LSR-T cells by G12D KRAS.

[0279] As described herein, the present disclosure provides, among other things, LSR- expressing T cells that are activated by proteins that are typically only found intracellularly. Moreover, by specifically targeting proteins that are mutated in cancer cells, a layer of specificity is introduced that is not attainable by simply targeting cell surface proteins. The KRAS-LSR construct used in the present Examples was designed to target G12D KRAS, a mutant form of KRAS commonly found in a number of tumors, in particular pancreatic cancers.

[0280] Prior to this experiment, there was only limited data available on specificity of the KRAS antibody, G12D KRAS, from which the KRAS-LSR was derived based on binding data. Specificity of a KRAS-LSR described herein was tested by incubating zlessMeso-CAR / KRAS-LSR-T cells with a series of KRAS mutant purified proteins. Protocol for IFN-gamma stimulation by KRAS mutant protein

[0281] 1x105human T cells previously transfected with zlessMeso-CAR / KRAS-LSR dual expression vector were seeded in each well of a 96 round bottom plate, in RPMI medium, at a volume of 50 µL per well. Page 75 of 89 12923515v1Docket No.: 2017422-0007

[0282] KRAS (Acro, KRS-H51H4) and KRAS G12A (Acro, KRS-H51H1), G12C (Acro, KRS-H51H3), G12R (Acro, KRS-H51H2), G12D (Acro, KRS-H51H6), G12S (Acro, KRS-H5144), G13D (Acro, KRS-H51H7), Q61H (Acro, KRS-H51H5) proteins were prepared in three final concentrations, 5 µg / mL, 1 µg / mL and 0.2 µg / mL and 50 µL added into the well containing 50 µL of target cells.

[0283] After 24 hours, the plate was spun at 350g for 2 minutes and the supernatant was collected for testing IFN-gamma levels by ELISA. Protocol for IFN-gamma ELISA

[0284] The 96 well plate was coated with 2.0 mg / mL anti-IFN-gamma NIB42 (BD Pharmingen, ThermoFisher, Cat# 551221) at 100 mL per well in 0.1 M carbonate at pH 9.5 overnight at 40C. The plate was blocked with 0.3% NF milk in TBS (200 mL / well) for 1 hour at room temperature and then washed three times with wash buffer at 200 mL / well (1x TBST: 0.1 M Tris, 0.5 M NaCl, 0.05% Tween20). The IFN-gamma standard (recombinant human IFN-gamma, ThermoFisher, Cat# RIFNG100) was prepared with starting concentration at 0.1 mg / mL with three times serial dilution and added at 100 mL per well. The 100 mL cell culture supernatant taken after 24 hours incubation was transferred to the plate. Both cell culture supernatant and the IFN-gamma standard in the wells were incubated for 1 hour at room temperature. The dilution buffer for the standard was 1% BSA in 1x TBS (0.1 M Tris, 0.5 M NaCl). The plate was washed three times with wash buffer. The biotinylated mouse anti-human IFN-gamma (BD Pharmingen, from ThermoFisher, Cat# 554550) was added at 1 mg / mL concentration (100 mL) per well at room temperature for 1 hour. The plate was then washed three times with wash buffer. HRP-conjugated SA (Pierce high sensitivity Streptavidin-HRP, Thermo Fisher, Cat# 21130) was added at 1:2000, 100 mL per well, and incubated at room temperature in the dark for 1 hour. The plate was then washed three timed with wash buffer.100 mL per well of 1-Step Ultra TMB-ELISA (ThermoFisher, Cat# 34028) was added to each well for color development and then 100 mL of ELISA stop solution (Invitrogen, Cat# SS04) was applied to each well. The plate was read at 450 nm and the IFN-gamma concentration was calculated based on the standard curve. Page 76 of 89 12923515v1Docket No.: 2017422-0007

[0285] As shown in FIG.29, activation of T cells expressing the zlessMeso- CAR / KRAS-LSR was highly dependent on the presence of G12D KRAS. As described herein, it is an insight of the present disclosure that lysis of tumor cells containing G12D KRAS would preferentially activate a KRAS-LSR, compared to non-tumor cells that contain wild-type KRAS.

[0286] Accordingly, the present Example documents specific activation of zlessMeso- CAR / KRAS-LSR-T cells by G12D KRAS. Those skilled in the art, reading the present Example, will appreciate that its findings can be expected to be applicable to LSRs targeting other antigens and / or to different CARs. Example 13: Exemplary zlessMeso-CAR and KRAS-LSR are Inactive When Independently Expressed

[0287] The present Example demonstrates that T cells (e.g., CAR-T cells) expressing a KRAS-targeting LSR as exemplified herein (referred to in the present Example as “KRAS- LSR-T cells”) show tumor-specific efficacy (e.g., increased tumor-specificity relative to comparable CAR-T cells not expressing the KRAS-LSR-T cells) without increasing non- specific activity. The present Example demonstrates that a KRAS-LSR, which recognizes an intracellular protein is also inactive when expressed independently from a Meso-CAR.

[0288] As described herein, it is an insight of the present disclosure that specificity of a CAR-T cell therapy can be improved by a) reducing the ability of CARs that target surface proteins to kill cells and b) by reactivating these CARs when a co-expressed LSR is activated by tumor-specific intracellular proteins. FIG.30 shows that a mesothelin- targeting CAR lacking a CD3^ (CD3Z) domain (zlessMeso-CAR) lacked cell killing activity. FIG.30 also shows that the KRAS-LSR, which recognizes an intracellular protein, is also inactive when expressed independently from a Meso-CAR. Cell killing is detected when KRAS-LSR-T cells are combined with Meso-CAR-T cells. Mixing of cells that independently express the zlessMeso-CAR, the Meso-CAR or the KRAS-LSR did not lead to increased cell killing.

[0289] Accordingly, the present Example documents tumor-specific efficacy of lysis sensing receptor and a CAR targeting a tumor cell surface antigen. The present Example documents tumor-specific efficacy of KRAS-LSR-T cells co-expressing a KRAS-LSR and a Page 77 of 89 12923515v1Docket No.: 2017422-0007 Meso-CAR. Those skilled in the art, reading the present Example, will appreciate that its findings can be expected to be applicable to LSRs targeting other antigens and / or to different CARs. Example 14: Anti-tumor Activity of Exemplary zlessCAR / LSR-T cells is Activated by Priming Tumor Cell Killing

[0290] The present Example demonstrates that anti-tumor activity of a zlessCAR / LSR cell (e.g., zlessCAR / LSR-T cell) can be activated by priming tumor cell killing.

[0291] SW1116 cells were cultured as described in Example 8. Wells of the plate were coated with a solution containing 3.2 µg collagen (Sigma C8919) for 7 hours then dried overnight before the addition of SW1116 cells.

[0292] In one exemplary embodiment, anti-tumor activity of a zlessCAR / LSR-T cell can be activated by priming tumor cell killing. Once primed, zlessCAR / LSR-T cells would specifically target tumor cells as lysis-associated antigens would be released from cancer cells and not be released from any healthy cells that may have been lysed during a priming event. As described herein, neither zlessMeso-CAR T cells nor KRAS-LSR-T cells were able to kill tumor cells on their own. Furthermore mixing of these two type of T cells had no cell killing effect (see, FIG.30).

[0293] Co-expression of both a zlessMeso-CAR and a KRAS-LSR in the same cell stopped growth of SW1116 cells, mesothelin-expressing, G12D KRAS human colorectal cells, but does not cause cell killing. Without wishing to be bound to any theory, the present disclosure notes that lack of cell killing is due to inability of the KRAS-LSR to be activated by intracellular G12D KRAS. Release of G12D KRAS was primed by the addition of a small amount of fully active Meso-CAR-T cells (1:10). The amount of Meso-CAR-T added as a primer was insufficient to lead to significant cell killing on its own. The combination of zlessMeso-CAR / KRAS-LSR-T cells (3:1) and Meso-CAR-T cells (1:10) led to cell killing. Priming with a higher ratio of Meso-CAR-T cells (1:1) also led to enhanced cell killing when combined with Meso-CAR / KRAS-LSR-T cells (3:1). The zlessMeso-CAR / KRAS- LSR-T cells (3:1) and Meso-CAR-T cells (1:1) showed earlier and higher rate of cell killing relative to cell killing observed with Meso-CAR-T primer cells alone (see, FIG.31). Page 78 of 89 12923515v1Docket No.: 2017422-0007

[0294] In the present Example, SW1116 cells, having a G12D KRAS mutation, and ASPC-1 cells, having wild-type KRAS, were used to demonstrate ability of a KRAS-LSR-T cell provided herein to be specifically activated when targeting cells that contain G12D KRAS. Expression of IFN-gamma is known to be correlated with cytotoxicity of CAR-T cells (Pattu et al., International Journal of Molecular Sciences.2025; 26(14):7024; the contents of which is hereby incorporated by reference herein in its entirety). In this Example, IFN-gamma secretion was used as a measure of T cell activation and a surrogate for CAR-T cell mediated cell killing of target cells.

[0295] Both SW1116 and APC-1 express mesothelin and were killed by T cells expressing a mesothelin-targeting CAR (e.g., Meso-CAR). A low basal level of IFN-gamma is observed in the absence of Meso-CAR-T cells or in the presence of a low level of Meso- CAR-T cells (1:10). At a ratio of 1 Meso-CAR-T cell to 1 target cell (SW1116 or ASPC-1), a high level of IFN-gamma production with each of the target cell lines was observed when measured after 25 hours. The zlessMeso-CAR / KRAS-LSR-T cells at a 3:1 E:T ratio led to secretion of IFN-gamma that was about 40% of the maximum observed when incubated with SW1116 cells. Addition of a low level of Meso-CAR-T cells (1:10) to zlessMeso- CAR / KRAS-LSR-T cells (3:1) led to increased IFN-gamma secretion, consistent with ability of Meso-CAR-T cells to prime zlessMeso-CAR / KRAS-LSR-T cell killing by releasing KRAS G12D from a small number of cells. Such increase in IFN-gamma expression was not observed in the absence of zlessMeso-CAR / KRAS-LSR-T cells. A lower amount of IFN-gamma expression and stimulation by Meso-CAR-T cell priming was observed with ASPC-1 cells, consistent with a preferential stimulation of the KRAS LSR by G12D KRAS versus wild-type KRAS (see, FIG.32).

[0296] Accordingly, the present Example documents increased anti-tumor activity of a zlessCAR / LSR cell (e.g., zlessCAR / LSR-T cell) can be activated by priming tumor cell killing. In particular, the present Example demonstrates that anti-tumor activity of zlessMeso-CAR / KRAS-LSR-T cells is increased with priming of tumor cell killing by addition of Meso-CAR-T cells, as shown by IFN-gamma secretion, relative to activity of zlessMeso-CAR / KRAS-LSR-T cells alone. Page 79 of 89 12923515v1Docket No.: 2017422-0007 EXEMPLARY EMBODIMENTS

[0297] Embodiment 1. A population of T cells engineered to express: (1) an engineered lysis sensor receptor (LSR) comprising: a) a lysis-associated-antigen binding moiety; b) a transmembrane element; and c) a signaling element (e.g., a T cell receptor co-stimulation element); and (2) a chimeric antigen receptor (CAR) comprising: (a) an antigen-binding moiety; (b) a transmembrane element; and (c) a signaling element (e.g., a T cell receptor co-stimulation element), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element.

[0298] Embodiment 2. The population of T cells of embodiment 1, wherein the population of T cells is isolated from a human.

[0299] Embodiment 3. The population of T cells of embodiment 2, wherein the human is a patient to whom the engineered population of T cells is to be administered.

[0300] Embodiment 4. The population of T cells of embodiment 1, wherein the T cells are obtained from a site that is in the vicinity of a tumor.

[0301] Embodiment 5. The population of T cells of embodiment 4, wherein the site comprises part or all of the tumor.

[0302] Embodiment 6. A method of producing a population of T cells according to any of the preceding claims comprising steps of: (A) obtaining a plurality of T cells; and Page 80 of 89 12923515v1Docket No.: 2017422-0007 (B) introducing into the T cells: (1) a CRISPR / Cas gene editing system comprising, (i) a gRNA molecule having a nucleotide sequence comprising a targeting sequence specific to an integration site in the T cell genome, and (ii) a Cas9 protein or a nucleic acid encoding a Cas9 protein; and (2) a virion comprising a viral capsid and one or more polynucleotide constructs encoding an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element, so that a population of engineered T cells is produced.

[0303] Embodiment 7. A method of producing a population of engineered T cells according to any of the preceding embodiments comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises one or more polynucleotide constructs encoding an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element.

[0304] Embodiment 8. A method of treating a disease, comprising a step of: (a) administering to a subject suffering from a disease, disorder or condition, a population of T cells engineered to express an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element that responds to / targets an intracellular product of diseased cell(s) in the subject.

[0305] Embodiment 9. The method of embodiment 8, wherein the LSR responds to / targets an intracellular product of diseased cell(s) in the subject and the CAR responds to / targets an extracellular product of diseased cell(s) in the subject.

[0306] Embodiment 10. The method of embodiment 9, wherein the CAR responds to / targets a surface-expressed antigen in the subject. Page 81 of 89 12923515v1Docket No.: 2017422-0007

[0307] Embodiment 11. The method of treatment of embodiment 8, wherein the subject has a disease associated with expression of a tumor or disease-related antigen (e.g., a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the disease-related antigen).

[0308] Embodiment 12. The method of embodiment 8, wherein the diseased cells are tumor cells.

[0309] Embodiment 13. The method of embodiment 8, wherein the diseased cells are infected cells.

[0310] Embodiment 14. The method of embodiment 8, wherein the step of administering comprises administering a population of T cells engineered by a process comprising steps of: (a) obtaining a plurality of T cells; (b) transducing the cells with a preparation of virions comprising a viral capsid and one or more polynucleotide constructs encoding an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element, optionally further selecting or screening for the transduced cells; and (c) administering the transduced cells to a subject in need thereof.

[0311] Embodiment 15. The method of treatment of embodiment 8, wherein the method of treatment comprises at least two successive doses.

[0312] Embodiment 16. The method of treatment of embodiment 15, wherein the second dose further comprises an indication that a disease, disorder or condition in a subject has progressed.

[0313] Embodiment 17. The method of treatment of embodiment 8, wherein the treatment is administered in combination with another immune oncology therapy. Page 82 of 89 12923515v1Docket No.: 2017422-0007

[0314] Embodiment 18. The method of treatment of embodiment 17, wherein the immune oncology therapy is a checkpoint inhibitor, a cytotoxic chemotherapy, or radiation therapy.

[0315] Embodiment 19. The method of embodiment 17, wherein the immune oncology therapy is or comprises infusion with a lysis-associated antigen.

[0316] Embodiment 20. A method of stimulating endogenous tumor infiltrating lymphocytes (TIL) in a subject, the method comprising steps of: (a) obtaining a population of TIL from a subject; and (b) engineering the population of T cells to express an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ zeta (e.g., CD3 zeta, CD3Z, CD3C) activating element, so that a population of engineered TIL is generated.

[0317] Embodiment 21. The method of embodiment 20, the method further comprising a step of: administering the population of engineered TIL.

[0318] Embodiment 22. The method of embodiment 20, wherein the obtained population of TIL comprises a plurality of TIL having T cell receptors (TCRs) directed to different antigens.

[0319] Embodiment 23. The method of embodiment 20, wherein the administered population of engineered TIL comprises a plurality of engineered TIL having T cell receptors (TCRs) directed to different antigens.

[0320] Embodiment 24. A method of manufacturing a population of T cells comprising: (i) engineering the population of T cells to express an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling Page 83 of 89 12923515v1Docket No.: 2017422-0007 element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element.

[0321] Embodiment 25. The method of manufacturing of embodiment 24, further comprising a step of isolating T cells from a human subject and administering the isolated T cells to the human subject. EQUIVALENTS

[0322] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims: Page 84 of 89 12923515v1

Claims

Docket No.: 2017422-0007 CLAIMS We claim:

1. A population of T cells engineered to express: (1) an engineered lysis sensor receptor (LSR) comprising: a) a lysis-associated-antigen binding moiety; b) a transmembrane element; and c) a signaling element (e.g., a T cell receptor co-stimulation element); and (2) a chimeric antigen receptor (CAR) comprising: (a) an antigen-binding moiety; (b) a transmembrane element; and (c) a signaling element (e.g., a T cell receptor co-stimulation element), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element.

2. The population of T cells of claim 1, wherein the population of T cells is isolated from a human.

3. The population of T cells of claim 2, wherein the human is a patient to whom the engineered population of T cells is to be administered.

4. The population of T cells of claim 1, wherein the T cells are obtained from a site that is in the vicinity of a tumor.

5. The population of T cells of claim 4, wherein the site comprises part or all of the tumor.

6. A method of producing a population of T cells according to any of the preceding claims comprising steps of: (a) obtaining a plurality of T cells; and (b) introducing into the T cells: (1) a CRISPR / Cas gene editing system comprising, Page 85 of 89 12923515v1Docket No.: 2017422-0007 (i) a gRNA molecule having a nucleotide sequence comprising a targeting sequence specific to an integration site in the T cell genome, and (ii) a Cas9 protein or a nucleic acid encoding a Cas9 protein; and (2) a virion comprising a viral capsid and one or more polynucleotide constructs encoding an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element, so that a population of engineered T cells is produced.

7. A method of producing a population of engineered T cells according to any of the preceding claims comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises one or more polynucleotide constructs encoding an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element.

8. A method of treating a disease, comprising a step of: (a) administering to a subject suffering from a disease, disorder or condition, a population of T cells engineered to express an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element that responds to / targets an intracellular product of diseased cell(s) in the subject.

9. The method of claim 8, wherein the LSR responds to / targets an intracellular product of diseased cell(s) in the subject and the CAR responds to / targets an extracellular product of diseased cell(s) in the subject.

10. The method of claim 9, wherein the CAR responds to / targets a surface-expressed antigen in the subject.

11. The method of treatment of claim 8, wherein the subject has a disease associated with expression of a tumor or disease-related antigen (e.g., a proliferative disease, a Page 86 of 89 12923515v1Docket No.: 2017422-0007 precancerous condition, a cancer, and a non-cancer related indication associated with expression of the disease-related antigen).

12. The method of claim 8, wherein the diseased cells are tumor cells.

13. The method of claim 8, wherein the diseased cells are infected cells.

14. The method of claim 8, wherein the step of administering comprises administering a population of T cells engineered by a process comprising steps of: (a) obtaining a plurality of T cells; (b) transducing the cells with a preparation of virions comprising a viral capsid and one or more polynucleotide constructs encoding an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element, optionally further selecting or screening for the transduced cells; and (c) administering the transduced cells to a subject in need thereof.

15. The method of treatment of claim 8, wherein the method of treatment comprises at least two successive doses.

16. The method of treatment of claim 15, wherein the second dose further comprises an indication that a disease, disorder or condition in a subject has progressed.

17. The method of treatment of claim 8, wherein the treatment is administered in combination with another immune oncology therapy.

18. The method of treatment of claim 17, wherein the immune oncology therapy is a checkpoint inhibitor, a cytotoxic chemotherapy, or radiation therapy.

19. The method of claim 17, wherein the immune oncology therapy is or comprises infusion with a lysis-associated antigen. Page 87 of 89 12923515v1Docket No.: 2017422-0007 20. A method of stimulating endogenous tumor infiltrating lymphocytes (TIL) in a subject, the method comprising steps of: (a) obtaining a population of TIL from a subject; and (b) engineering the population of T cells to express an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ zeta (e.g., CD3 zeta, CD3Z, CD3C) activating element, so that a population of engineered TIL is generated.

21. The method of claim 20, the method further comprising a step of: administering the population of engineered TIL.

22. The method of claim 20, wherein the obtained population of TIL comprises a plurality of TIL having T cell receptors (TCRs) directed to different antigens.

23. The method of claim 20, wherein the administered population of engineered TIL comprises a plurality of engineered TIL having T cell receptors (TCRs) directed to different antigens.

24. A method of manufacturing a population of T cells comprising: (i) engineering the population of T cells to express an engineered lysis sensor receptor (LSR) and a chimeric antigen receptor (CAR), wherein the signaling element of the CAR does not comprise a CD3^ (e.g., CD3 zeta, CD3Z, CD3C) activating element.

25. The method of manufacturing of claim 24, further comprising a step of isolating T cells from a human subject and administering the isolated T cells to the human subject. Page 88 of 89 12923515v1

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